Detection of nucleic acid hybrids

ABSTRACT

Processes are disclosed using the depolymerization of a nucleic acid hybrid to qualitatively and quantitatively analyze for the presence of a predetermined nucleic acid. Applications of those processes include the detection of single nucleotide polymorphisms, identification of single base changes, speciation, determination of viral load, genotyping, medical marker diagnostics, and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/252,436,filed on Feb. 18, 1999, which is a continuation-in-part of U.S. Ser. No.09/042,287, filed Mar. 13, 1998, both of which are incorporated hereinby reference.

FIELD OF THE INVENTION

The invention relates to nucleic acid detection. More specifically, theinvention relates to the detection of targeted, predetermined nucleicacid sequences in nucleic acid target hybrids, and the variousapplications of their detection.

BACKGROUND OF THE INVENTION

Methods to detect nucleic acids and to detect specific nucleic acidsprovide a foundation upon which the large and rapidly growing field ofmolecular biology is built. There is constant need for alternativemethods and products. The reasons for selecting one method over anotherare varied, and include a desire to avoid radioactive materials, thelack of a license to use a technique, the cost or availability ofreagents or equipment, the desire to minimize the time spent or thenumber of steps, the accuracy or sensitivity for a certain application,the ease of analysis, or the ability to automate the process.

The detection of nucleic acids or specific nucleic acids is often aportion of a process rather than an end in itself. There are manyapplications of the detection of nucleic acids in the art, and newapplications are always being developed. The ability to detect andquantify nucleic acids is useful in detecting microorganisms, virusesand biological molecules, and thus affects many fields, including humanand veterinary medicine, food processing and environmental testing.Additionally, the detection and/or quantification of specificbiomolecules from biological samples (e.g. tissue, sputum, urine, blood,semen, saliva) has applications in forensic science, such as theidentification and exclusion of criminal suspects and paternity testingas well as medical diagnostics.

Some general methods to detect nucleic acids are not dependent upon apriori knowledge of the nucleic acid sequence. A nucleic acid detectionmethod that is not sequence specific, but is RNA specific is describedin U.S. Pat. No. 4,735,897, where RNA is depolymerized using apolynucleotide phosphorylase (PNP) in the presence of phosphate or usinga ribonuclease. PNP stops depolymerizing when a double-stranded RNAsegment is encountered, sometimes as the form of secondary structure ofsingle-stranded RNA, as is common in ribosomal RNA, transfer RNA, viralRNA, and the message portion of mRNA. PNP depolymerization of thepolyadenylated tail of mRNA in the presence of inorganic phosphate formsADP. Alternatively, depolymerization using a ribonuclease forms AMP. Theformed AMP is converted to ADP with myokinase, and ADP is converted intoATP by pyruvate kinase or creatine phosphokinase. Either the ATP or thebyproduct from the organophosphate co-reactant (pyruvate or creatine) isdetected as an indirect method of detecting mRNA.

In U.S. Pat. No. 4,735,897, ATP is detected by a luciferase detectionsystem. In the presence of ATP and oxygen, luciferase catalyzes theoxidation of luciferin, producing light that can then be quantifiedusing a luminometer. Additional products of the reaction are AMP,pyrophosphate and oxyluciferin.

Duplex DNA can be detected using intercalating dyes such as ethidiumbromide. Such dyes are also used to detect hybrid formation.

Hybridization methods to detect nucleic acids are dependent uponknowledge of the nucleic acid sequence. Many known nucleic aciddetection techniques depend upon specific nucleic acid hybridization inwhich an oligonucleotide probe is hybridized or annealed to nucleic acidin the sample or on a blot, and the hybridized probes are detected.

A traditional type of process for the detection of hybridized nucleicacid uses labeled nucleic acid probes to hybridize to a nucleic acidsample. For example, in a Southern blot technique, a nucleic acid sampleis separated in an agarose gel based on size and affixed to a membrane,denatured, and exposed to the labeled nucleic acid probe underhybridizing conditions. If the labeled nucleic acid probe forms a hybridwith the nucleic acid on the blot, the label is bound to the membrane.Probes used in Southern blots have been labeled with radioactivity,fluorescent dyes, digoxygenin, horseradish peroxidase, alkalinephosphatase and acridinium esters.

Another type of process for the detection of hybridized nucleic acidtakes advantage of the polymerase chain reaction (PCR). The PCR processis well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159). To briefly summarize PCR, nucleic acid primers,complementary to opposite strands of a nucleic acid amplification targetsequence, are permitted to anneal to the denatured sample. A DNApolymerase (typically heat stable) extends the DNA duplex from thehybridized primer. The process is repeated to amplify the nucleic acidtarget. If the nucleic acid primers do not hybridize to the sample, thenthere is no corresponding amplified PCR product. In this case, the PCRprimer acts as a hybridization probe. PCR-based methods are of limiteduse for the detection of nucleic acid of unknown sequence.

In a PCR method, the amplified nucleic acid product may be detected in anumber of ways, e.g. incorporation of a labeled nucleotide into theamplified strand by using labeled primers. Primers used in PCR have beenlabeled with radioactivity, fluorescent dyes, digoxygenin, horseradishperoxidase, alkaline phosphatase, acridinium esters, biotin and jackbean urease. PCR products made with unlabeled primers may be detected inother ways, such as electrophoretic gel separation followed by dye-basedvisualization.

Fluorescence techniques are also known for the detection of nucleic acidhybrids. U.S. Pat. No. 5,691,146 describes the use of fluorescenthybridization probes that are fluorescence-quenched unless they arehybridized to the target nucleic acid sequence. U.S. Pat. No. 5,723,591describes fluorescent hybridization probes that arefluorescence-quenched until hybridized to the target nucleic acidsequence, or until the probe is digested. Such techniques provideinformation about hybridization, and are of varying degrees ofusefulness for the determination of single base variances in sequences.Some fluorescence techniques involve digestion of a nucleic acid hybridin a 5′→3′ direction to release a fluorescent signal from proximity to afluorescence quencher, for example, TaqMan® (Perkin Elmer; U.S. Pat.Nos. 5,691,146 and 5,876,930).

Enzymes having template-specific polymerase activity for which some3′→5′ depolymerization activity has been reported include E. coli DNAPolymerase (Deutscher and Kornberg, J. Biol. Chem., 244(11):3019-28(1969)), T7 DNA Polymerase (Wong et al., Biochemistry 30:526-37 (1991);Tabor and Richardson, J. Biol. Chem. 265: 8322-28 (1990)), E. coli RNApolymerase (Rozovskaya et al., Biochem. J. 224:645-50 (1994)), AMV andRLV reverse transcriptases (Srivastava and Modak, J. Biol. Chem. 255:2000-4 (1980)), and HIV reverse transcriptase (Zinnen et al., J. Biol.Chem. 269:24195-202 (1994)). A template-dependent polymerase for which3′ to 5′ exonuclease activity has been reported on a mismatched end of aDNA hybrid is phage 29 DNA polymerase (de Vega, M. et al. EMBO J.,15:1182-1192, 1996)

A variety of methodologies currently exist for the detection of singlenucleotide polymorphisms (SNPS) that are present in genomic DNA. SNPsare DNA point mutations or insertions/deletions that are present atmeasurable frequencies in the population. SNPs are the most commonvariations in the genome. SNPs occur at defined positions within genomesand can be used for gene mapping, defining population structure, andperforming functional studies. SNPs are useful as markers because manyknown genetic diseases are caused by point mutations andinsertions/deletions.

In rare cases where an SNP alters a fortuitous restriction enzymerecognition sequence, differential sensitivity of the amplified DNA tocleavage can be used for SNP detection. This technique requires that anappropriate restriction enzyme site be present or introduced in theappropriate sequence context for differential recognition by therestriction endonuclease. After amplification, the products are cleavedby the appropriate restriction endonuclease and products are analyzed bygel electrophoresis and subsequent staining. The throughput of analysisby this technique is limited because samples require processing, gelanalysis, and significant interpretation of data before SNPs can beaccurately determined.

Single strand conformational polymorphism (SSCP) is a second techniquethat can detect SNPs present in an amplified DNA segment (Hayashi, K.Genetic Analysis: Techniques and Applications 9:73-79, 1992). In thismethod, the double stranded amplified product is denatured and then bothstrands are allowed to reanneal during electrophoresis in non-denaturingpolyacrylamide gels. The separated strands assume a specific foldedconformation based on intramolecular base pairing. The electrophoreticproperties of each strand are dependent on the folded conformation. Thepresence of single nucleotide changes in the sequence can cause adetectable change in the conformation and electrophoretic migration ofan amplified sample relative to wild type samples, allowing SNPs to beidentified. In addition to the limited throughput possible by gel-basedtechniques, the design and interpretation of SSCP based experiments canbe difficult. Multiplex analysis of several samples in the same SSCPreaction is extremely challenging. The sensitivity required in mutationdetection and analysis has led most investigators to use radioactivelylabeled PCR products for this technique.

In the amplification refractory mutation system (ARMS, also known asallele specific PCR or ASPCR), two amplification reactions are used todetermine if a SNP is present in a DNA sample (Newton et al. Nucl AcidsRes 17:2503, 1989; Wu et al. PNAS 86:2757, 1989). Both amplificationreactions contain a common primer for the target of interest. The firstreaction contains a second primer specific for the wild type productwhich will give rise to a PCR product if the wild type gene is presentin the sample. The second PCR reaction contains a primer that has asingle nucleotide change at or near the 3′ end that represents the basechange that is present in the mutated form of the DNA. The secondprimer, in conjunction with the common primer, will only function in PCRif genomic DNA that contains the mutated form of genomic DNA is present.This technique requires duplicate amplification reactions to beperformed and analyzed by gel electrophoresis to ascertain if a mutatedform of a gene is present. In addition, the data must be manuallyinterpreted.

Single base extension (GBA®) is a technique that allows the detection ofSNPs by hybridizing a single strand DNA probe to a captured DNA target(Nikiforov, T. et al. Nucl Acids Res 22:4167-4175). Once hybridized, thesingle strand probe is extended by a single base with labeleddideoxynucleotides. The labeled, extended products are then detectedusing calorimetric or fluorescent methodologies.

A variety of technologies related to real-time (or kinetic) PCR havebeen adapted to perform SNP detection. Many of these systems areplatform based, and require specialized equipment, complicated primerdesign, and expensive supporting materials for SNP detection. Incontrast, the process of this invention has been designed as a modulartechnology that can use a variety of instruments that are suited to thethroughput needs of the end-user. In addition, the coupling ofluciferase detection sensitivity with standard oligonucleotide chemistryand well-established enzymology provides a flexible and open systemarchitecture. Alternative analytical detection methods, such as massspectroscopy, HPLC, and fluorescence detection methods can also be usedin the process of this invention, providing additional assayflexibility.

SNP detection using real-time amplification relies on the ability todetect amplified segments of nucleic acid as they are during theamplification reaction. Three basic real-time SNP detectionmethodologies exist: (i) increased fluorescence of double strand DNAspecific dye binding, (ii) decreased quenching of fluorescence duringamplification, and (iii) increased fluorescence energy transfer duringamplification (Wittwer, C. et al. Biotechniques 22:130-138, 1997). Allof these techniques are non-gel based and each strategy will be brieflydiscussed.

A variety of dyes are known to exhibit increased fluorescence inresponse to binding double stranded DNA. This property is utilized inconjunction with the amplification refractory mutation system describedabove to detect the presence of SNP. Production of wild type or mutationcontaining PCR products are continuously monitored by the increasedfluorescence of dyes such as ethidium bromide or SYBER Green as theybind to the accumulating PCR product. Note that dye binding is notselective for the sequence of the PCR product, and high non-specificbackground can give rise to false signals with this technique.

A second detection technology for real time PCR, known generally asexonuclease primers (TaqMan® probes), utilizes the 5′ exonucleaseactivity of thermostable polymerases such as Taq to cleave dual-labeledprobes present in the amplification reaction (Wittwer, C. et al.Biotechniques 22:130-138, 1997; Holland, P et al PNAS 88:7276-7280,1991). While complementary to the PCR product, the probes used in thisassay are distinct from the PCR primer and are dually-labeled with botha molecule capable of fluorescence and a molecule capable of quenchingfluorescence. When the probes are intact, intramolecular quenching ofthe fluorescent signal within the DNA probe leads to little signal. Whenthe fluorescent molecule is liberated by the exonuclease activity of Taqduring amplification, the quenching is greatly reduced leading toincreased fluorescent signal.

An additional form of real-time PCR also capitalizes on theintramolecular quenching of a fluorescent molecule by use of a tetheredquenching moiety. The molecular beacon technology utilizeshairpin-shaped molecules with an internally-quenched fluorophore whosefluorescence is restored by binding to a DNA target of interest (Kramer,R. et al. Nat. Biotechnol. 14:303-308, 1996). Increased binding of themolecular beacon probe to the accumulating PCR product can be used tospecifically detect SNPs present in genomic DNA.

A final general fluorescent detection strategy used for detection of SNPin real time utilizes synthetic DNA segments known as hybridizationprobes in conjunction with a process known as fluorescence resonanceenergy transfer (FRET) (Wittwer, C. et al. Biotechniques 22:130-138,1997; Bernard, P. et al. Am. J. Pathol. 153:1055-1061, 1998). Thistechnique relies on the independent binding of labeled DNA probes on thetarget sequence. The close approximation of the two probes on the targetsequence increases resonance energy transfer from one probe to theother, leading to a unique fluorescence signal. Mismatches caused bySNPs that disrupt the binding of either of the probes can be used todetect mutant sequences present in a DNA sample.

There is a need for highly sensitive, diagnostic applications that arecapable of determining the number of virus molecules present in a body(“viral load”). For example, the presence of viral particles in thecirculation system or in specific tissues is a means of monitoring theseverity of viral infection. Several methods are currently used in theart for determining viral load. U.S. Pat. No. 5,667,964 discloses amethod for the determination of the number of HIV-1 infected patientcells using reactive oxygen-intermediate generators. U.S. Pat. No.5,389,512 discloses a method for determining the relative amount of aviral nucleic acid segment in a sample using PCR.

G. Garinis et al., J. Clin. Lab. Anal. 13:122-5 (1999) compare thedetermination of viral load results using an enzyme-linked immunosorbantassay (ELISA), a recombinant immunoblot assay (RIBA), and a reversetranscriptase polymerase chain reaction method (RT-PCR) in the detectionof hepatitis C virus (HCV) infection in haemodialysis patients. Thequantitative hepatitis HCV RT-PCR assay had a detection level of about2,000 viral copies/mL serum. Holguin et al., Eur. J. Clin. Microbiol.Infect. Dis. 18:256-9 (1999) compare plasma HIV-1 RNA levels usingseveral commercially available assays, namely the second-generationHIV-1 branched DNA assay, the Nuclisens assay, the Amplicor® Monitorreverse transcriptase polymerase chain reaction assay, and theUltradirect Monitor. Differing values were noted in comparing resultsamong these various assays. Boriskin et al., Arch. Dis. Child. 80:132-6(1999) used a nested polymerase chain reaction to measure HIV-1 proviralDNA and CMV genomic DNA in peripheral blood leukocytes of childreninfected with HIV-1. There remains a need for a reliable means to detectand quantify viral load. There is a demand for methods to determineviral load when the quantities of viral particles are very low.

In summary, there is a need for alternative methods for the detection ofnucleic acid hybrids. There is a demand for highly sensitive methodsthat are useful for determining the presence or absence of specificnucleic acid sequences, for example methods to determine viral load thatare able to reliably detect as few as 10 copies of a virus present in abody, tissue, fluid, or other biological sample. There is a great demandfor such methods to determine the presence or absence of nucleic acidsequences that differ slightly from sequences that might otherwise bepresent. There is a great demand for methods to determine the presenceor absence of sequences unique to a particular species in a sample.There is also a great demand for methods that are more highly sensitivethan the known methods, highly reproducible and automatable.

It would be beneficial if another method were available for detectingthe presence of a sought-after, predetermined target nucleotide sequenceor allelic variant. It would also be beneficial if such a method wereoperable using a sample size of the microgram to picogram scale. Itwould further be beneficial if such a detection method were capable ofproviding multiple analyses in a single assay (multiplex assays). Thedisclosure that follows provides one such method.

BREIF SUMMARY OF THE INVENTION

A method of this invention is used to determine the presence or absenceof a predetermined (known) nucleic acid target sequence in a nucleicacid sample. Such a method utilizes an enzyme that can depolymerize the3′-terminus of an oligonucleotide probe hybridized to a nucleic acidtarget sequence to release one or more identifier nucleotides whosepresence can then be determined.

One embodiment of the invention contemplates a method for determiningthe presence or absence of a predetermined nucleic acid target sequencein a nucleic acid sample. Thus, the presence or absence of at least onepredetermined nucleic acid target sequence is sought to be determined.More than one such predetermined target sequence can also be present inthe sample being assayed, and the presence or absence of more than onepredetermined nucleic acid target sequence can be determined. Theembodiment comprises the following steps.

A treated sample is provided that may contain a predetermined nucleicacid target sequence hybridized with a nucleic acid probe that includesan identifier nucleotide in the 3′-terminal region. The treated sampleis admixed with a depolymerizing amount of an enzyme whose activity isto release one or more nucleotides from the 3′-terminus of a hybridizednucleic acid probe to form a treated reaction mixture. The treatedreaction mixture is maintained under depolymerizing conditions for atime period sufficient to permit the enzyme to depolymerize hybridizednucleic acid and release identifier nucleotides therefrom. The presenceof released identifier nucleotides is analyzed to obtain an analyticaloutput, the analytical output indicating the presence or absence of thenucleic acid target sequence. The analytical output obtained by varioustechniques as discussed herein.

An analytical output is obtained by analyzing for the presence orabsence of released identifier nucleotide. The analytical outputindicates the presence or absence of the nucleotide at the predeterminedregion, and, thereby, the presence or absence of a first nucleic acidtarget. The analytical output is obtained by various techniques asdiscussed herein.

It is contemplated that an analytical output of the methods of theinvention can be obtained in a variety of ways. The analytical outputcan be ascertained by luminescence spectroscopy. In some preferredembodiments, analysis for released 3′-terminal region indicatornucleotides comprises the detection of ATP, either by a luciferasedetection system (luminescence spectroscopy) or an NADH detection system(absorbance spectroscopy). In particularly preferred embodiments wheregreater sensitivity is desired, ATP molecules are formed by a phosphatetransferring step, for example using an enzyme such as NDPK in thepresence of ADP, from the nucleotide triphosphates produced by thedepolymerizing step. In some embodiments the ATP is amplified to form aplurality of ATP molecules. In the ATP detection embodiments, typicallythe enzyme (NDPK) for converting nucleotides and added ADP into ATP ispresent in the depolymerization reaction, and thus they are denoted as a“one pot”0 method.

In an alternative embodiment, the analytical output is obtained byfluorescence spectroscopy. It is contemplated that an identifiernucleotide includes a fluorescent label. An identifier nucleotide can befluorescently labeled prior to, or after, release of the identifiernucleotide. It is also contemplated that other than a releasedidentifier nucleotide contains a fluorescent tag. In such an embodiment,the release of nucleotides in a process of the invention is ascertainedby a determination of a difference in the length of the polynucleotideprobe, for example by capillary electrophoresis imaged by a fluorescenttag at the 5′ terminus of the probe or in a region other than the 3′terminal region.

In an alternative embodiment the analytical output is obtained by massspectrometry. It is preferred here that an identifier nucleotide be anucleotide analog or a labeled nucleotide and have a molecular mass thatis different from the mass of a usual form of that nucleotide, althougha difference in mass is not required. It is also noted that with afluorescently labeled identifier nucleotide, the analytical output canalso be obtained by mass spectrometry. It is also contemplated that theanalysis of released nucleotide be conducted by ascertaining thedifference in mass of the probe after a depolymerization step of aprocess of the invention.

In another alternative embodiment, the analytical output is obtained byabsorbance spectroscopy. Such analysis monitors the absorbance of lightin the ultraviolet and visible regions of the spectrum to determine thepresence of absorbing species. In one aspect of such a process, releasednucleotides are separated from hybridized nucleic acid and otherpolynucleotides by chromatography (e.g. HPLC or GC) or electrophoresis(e.g. PAGE or capillary electrophoresis). Either the released identifiernucleotide or the remainder of the probe can be analyzed for toascertain the release of the identifier nucleotide in a process of theinvention. In another aspect of such a process a label may beincorporated in the analyzed nucleic acid.

In a contemplated embodiment, a sample to be assayed is admixed with oneor more nucleic acid probes under hybridizing conditions to form ahybridization composition. The 3′-terminal region of the nucleic acidprobe hybridizes with partial or total complementarity to the nucleicacid target sequence when that sequence is present in the sample. The3′-terminal region of the nucleic acid probe includes an identifiernucleotide. The hybridization composition is maintained underhybridizing conditions for a time period sufficient to form a treatedsample that may contain said predetermined nucleic acid target sequencehybridized with a nucleic acid probe. The treated sample is admixed witha depolymerizing amount of an enzyme whose activity is to release one ormore nucleotides from the 3′-terminus of a hybridized nucleic acid probeto form a treated reaction mixture. The treated reaction mixture ismaintained under depolymerizing conditions for a timer period sufficientto permit the enzyme to depolymerize hybridized nucleic acid and releaseidentifier nucleotides therefrom. The presence of released identifiernucleotides is analyzed to obtain an analytical output, the analyticaloutput indicating the presence or absence of the nucleic acid targetsequence. The analytical output may be obtained by various techniques asdiscussed above.

One method of the invention contemplates interrogating the presence orabsence of a specific base in a nucleic acid target sequence in a sampleto be assayed, and comprises the following steps.

A hybridization composition is formed by admixing a sample to be assayedwith one or more nucleic acid probes under hybridizing conditions. Thesample to be assayed may contain a nucleic acid target sequence to beinterrogated. The nucleic acid target comprises at least one base whosepresence or absence is to be identified. The hybridization compositionincludes at least one nucleic acid probe that is substantiallycomplementary to the nucleic acid target sequence and comprises at leastone predetermined nucleotide at an interrogation position, and anidentifier nucleotide in the 3′-terminal region.

A treated sample is formed by maintaining the hybridization compositionunder hybridizing conditions for a time period sufficient for basepairing to occur when a probe nucleotide at an interrogation position isaligned with a base to be identified in the target sequence. A treatedreaction mixture is formed by admixing the treated sample with an enzymewhose activity is to release one or more identifier nucleotides from the3′-terminus of a hybridized nucleic acid probe to depolymerize thehybrid. The treated reaction mixture is maintained under depolymerizingconditions for a time period sufficient to permit the enzyme todepolymerize the hybridized nucleic acid and release an identifiernucleotide.

An analytical output is obtained by analyzing for the presence orabsence of released identifier nucleotides. The analytical outputindicates the presence or absence of the specific base or bases to beidentified. The analytical output is obtained by various techniques, asdiscussed herein. Preferably, an identifier nucleotide is at theinterrogation position.

In one aspect of a method of the invention, the nucleic acid targetsequence is selected from the group consisting of deoxyribonucleic acidand ribonucleic acid. The method that identifies the particular basepresent at an interrogation position, optionally comprises a firstprobe, a second probe, a third probe, and a fourth probe. Aninterrogation position of the first probe comprises a nucleic acidresidue that is a deoxyadenosine or adenosine residue. An interrogationposition of the second probe comprises a nucleic acid residue that is adeoxythymidine or uridine residue. An interrogation position of thethird probe comprises a nucleic acid residue that is a deoxyguanosine orguanosine residue. An interrogation position of the fourth nucleic acidprobe comprises a nucleic acid residue that is a deoxycytosine orcytosine residue.

In another aspect of the invention, the sample containing a plurality oftarget nucleic acid sequences is admixed with a plurality of the nucleicacid probes. Several analytical outputs can be obtained from suchmultiplexed assays. In a first embodiment, the analytical outputobtained when at least one nucleic acid probes hybridizes with partialcomplementarity to one target nucleic acid sequence is greater than theanalytical output when all of the nucleic acid probes hybridize withtotal complementarity to their respective nucleic acid target sequences.In a second embodiment, the analytical output obtained when at least onenucleic acid probe hybridizes with partial complementarity to one targetnucleic acid sequence is less than the analytical output when all of thenucleic acid probes hybridize with total complementarity to theirrespective nucleic acid target sequences. In a third embodiment, theanalytical output obtained when at least one nucleic acid probehybridizes with total complementarity to one nucleic acid targetsequence is greater than the analytical output when all of the nucleicacid probes hybridize with partial complementarity to their respectivenucleic acid target sequences. In a fourth embodiment, the analyticaloutput obtained when at least one nucleic acid probe hybridizes withtotal complementarity to one target nucleic acid sequence is less thanthe analytical output when all of the nucleic acid probes hybridize withpartial complementarity to their respective nucleic acid targetsequences. The depolymerizing enzymes are as described herein.

Yet another embodiment of the invention contemplates a method fordetermining the presence or absence of a first nucleic acid target in anucleic acid sample that may contain that target or may contain asubstantially identical second target. For example, the second targetmay have a base substitution, deletion or addition relative to the firstnucleic acid target. This embodiment comprises the following steps.

A sample to be assayed is admixed with one or more nucleic acid probesunder hybridizing conditions to form a hybridization composition. Thefirst and second nucleic acid targets each comprise a region of sequenceidentity except for at least a single nucleotide at a predeterminedposition that differs between the targets. The nucleic acid probe issubstantially complementary to the nucleic acid target region ofsequence identity and comprises at least one nucleotide at aninterrogation position. An interrogation position of the probe isaligned with the predetermined position of a target when a target andprobe are hybridized. The probe also includes an identifier nucleotidein the 3′-terminal region.

The hybridization composition is maintained under hybridizing conditionsfor a time period sufficient to form a treated sample wherein thenucleotide at the interrogation position of the probe is aligned withthe nucleotide at the predetermined position in the region of identityof the target.

A treated reaction mixture is formed by admixing the treated sample witha depolymerizing amount of an enzyme whose activity is to release one ormore nucleotides from the 3′-terminus of a hybridized nucleic acidprobe. The reaction mixture is maintained under depolymerizationconditions for a time period sufficient to permit the enzyme todepolymerize the hybridized nucleic acid and release the identifiernucleotide.

An analytical output is obtained by analyzing for the presence orabsence of released identifier nucleotides. The analytical outputindicates the presence or absence of the nucleotide at the predeterminedregion, and; thereby, the presence or absence of a first nucleic acidtarget.

One aspect of the above method is comprised of a first probe and asecond probe. The first probe comprises a nucleotide an interrogationposition that is complementary to a first nucleic acid target at apredetermined position. The second probe comprises a nucleotide at aninterrogation position that is complementary to a second nucleic acidtarget at a predetermined position.

In one aspect of a process of the invention, the depolymerizing enzyme,whose activity is to release nucleotides, is a template-dependentpolymerase, whose activity is to depolymerize hybridized nucleic acidwhose 3′-terminal nucleotide is matched, in the 3′→5′ direction in thepresence of pyrophosphate ions to release one or more nucleotides. Thus,the enzyme's activity is to depolymerize hybridized nucleic acid torelease nucleotides under depolymerizing conditions. Preferably, thisenzyme depolymerizes hybridized nucleic acids whose bases in the3′-terminal region of the probe are matched with total complementarityto the corresponding bases of the nucleic acid target. The enzyme willcontinue to release properly paired bases from the 3′-terminal regionand will stop when the enzyme arrives at a base that is mismatched.

In an alternative aspect of the process (method), the depolymerizingenzyme, whose activity is to release nucleotides, exhibits a 3′→5′exonuclease activity in which hybridized nucleic acids having one ormore mismatched bases at the 3′-terminus of the hybridized probe aredepolymerized. Thus, the enzyme's activity is to depolymerize hybridizednucleic acid to release nucleotides under depolymerizing conditions. Inthis embodiment, the hybrid may be separated from the free probe priorto enzyme treatment. In some embodiments, an excess of target may beused so that the concentration of free probe in the enzyme reaction isextremely low.

In still another alternative aspect of a process of the invention, thedepolymerizing enzyme exhibits a 3′ to 5′ exonuclease activity on adouble-stranded DNA substrate having one or more matched bases at the 3′terminus of the hybrid. The enzyme's activity is to depolymerizehybridized nucleic acid to release nucleotides containing a 5′ phosphateunder depolymerizing conditions.

In particularly preferred embodiments where greater sensitivity isdesired, ATP molecules are formed by a phosphate transferring step,(e.g. using NDPK in the presence of ADP), from the dNTPs produced by thedepolymerizing step. In some embodiments, the ATP can be amplified toform a plurality of ATP molecules.

In one aspect of the invention, the nucleic acid sample to be assayed isobtained from a biological sample that is a solid or liquid. Exemplarysolid biological samples include animal tissues such as those obtainedby biopsy or post mortem, and plant tissues such as leaves, roots,stems, fruit and seeds. Exemplary liquid samples include body fluidssuch as sputum, urine, blood, semen and saliva of an animal, or a fluidsuch as sap or other liquid obtained when plant tissues are cut or plantcells are lysed or crushed. In one aspect of the method, thepredetermined nucleic acid target sequence is a microbial or viralnucleic acid.

In some preferred embodiments of the invention, the predeterminednucleic acid target sequence is a viral nucleic acid. Viral load, theamount of virus present, can be determined from the magnitude of theanalytical output from a predetermined amount of biological sample suchas animal fluid or tissue. In some preferred embodiments, the presenceor absence of a mutation in the viral genome can be determined.

In another aspect of the method, the nucleic acid sample is obtainedfrom a food source. In one process of the method, the food source is aplant or is derived from plant material, and the predetermined nucleicacid target sequence is a sequence not native to that plant. In oneaspect of the method, the nucleic acid sequence not native to thesubject plant is a transcription control sequence. In one preferredembodiment of the invention, the transcription control sequence is the35S promoter or the NOS terminator, or both. In another aspect of themethod, the predetermined nucleic acid target sequence in a food sourcesample is a sequence native to the plant.

Another embodiment of the invention contemplates a method forselectively detecting poly(A)⁺ RNA, and comprises the following steps. Ahybridization composition is formed by admixing, under hybridizingconditions, a sample to be assayed with an oligo(dT) probe including a3′ terminal region identifier nucleotide. The hybridization compositionis maintained under hybridizing conditions for a time period sufficientto form a treated sample that contains the poly(A)⁺ RNA target sequencehybridized with the oligo(dT) probe. A treated reaction mixture isformed by admixing the treated sample with an enzyme whose activity isto release one or more identifier nucleotides from the 3′-terminalregion of the nucleic acid hybrid. The reaction mixture is maintainedunder depolymerizing conditions for a time period sufficient to permitthe enzyme to depolymerize the hybridized nucleic acid and release anidentifier nucleotide. An analytical output is obtained by analyzing thereaction mixture for the presence of released identifier nucleotide. Theanalytical output indicates the presence of poly(A)⁺ RNA.

A further embodiment of the invention, such as is used for Single TandemRepeat (STR) detection, contemplates a method for determining the numberof known sequence repeats that are present in a nucleic acid targetsequence in a nucleic acid sample. A method for determining the numberof known sequence repeats comprises the following steps. A plurality ofseparate treated samples is provided. Each treated sample contains anucleic acid target sequence hybridized with a nucleic acid probe. Thenucleic acid target sequence contains a plurality of known sequencerepeats and a downstream non-repeated region. Each nucleic acid probecontains a different number of complementary repeats of the knownsequence, an identifier nucleotide in the 3′-terminal region and a5′-terminal locker sequence. The 5′-terminal locker sequence iscomplementary to the downstream non-repeated region of the target andcomprises 1 to about 20 nucleotides, preferably 5 to 20 nucleotides,most preferably 10 to 20 nucleotides. The various probes representcomplements to possible alleles of the target nucleic acid. A treateddepolymerization reaction mixture is formed by admixing each treatedsample with a depolymerizing amount of an enzyme whose activity is torelease one or more nucleotides from the 3′-terminus of a hybridizednucleic acid probe. The treated depolymerization reaction mixture ismaintained under depolymerizing conditions for a time period sufficientto permit the enzyme to depolymerize the hybridized nucleic acid probeand release an identifier nucleotide. The samples are analyzed for thepresence or absence of released identifier nucleotide to obtain ananalytical output. The analytical output from the sample whose probecontained the same number of sequence repeats as present in the targetnucleic acid is indicative of and determines the number of sequencerepeats present in the nucleic acid target.

In one aspect of the method, the nucleic acid sample contains twonucleic acid targets representing alleles, and is homozygous withrespect to the number of known sequence repeats of the two alleles. Inan alternative method of the invention, the nucleic acid sample isheterozygous with respect to the two alleles. In another method of theinvention, an identifier nucleotide is a nucleotide that is part of theregion containing a repeated sequence. In an alternative method of theinvention, an identifier nucleotide of the probe sequence is part of theregion containing a non-repeating sequence that is complementary to thatlocated in the target nucleic acid 5′ to the repeated known sequence. Inthis latter aspect of the method, the identifier nucleotide is presentin a sequence containing 1 to about 20 nucleic acids that iscomplementary to a non-repeating sequence of the target nucleic acidlocated in the probe 3′ to the known sequence repeats. The repeatedknown sequence present in a nucleic acid target sequence typically has alength of 2 to about 24 bases per repeat.

A further embodiment of the invention contemplates a method usingthermostable DNA polymerase as a depolymerizing enzyme for determiningthe presence or absence of a at least one predetermined nucleic acidtarget sequence in a nucleic acid sample, and comprises the followingsteps.

A treated sample is provided that may contain a predetermined nucleicacid target sequence hybridized to a nucleic acid probe whose3′-terminal region is complementary to the predetermined nucleic acidtarget sequence and includes an identifier nucleotide in the 3′-terminalregion. A treated depolymerization reaction mixture is formed byadmixing a treated sample with a depolymerizing amount of a enzyme whoseactivity is to release an identifier nucleotide from the 3′-terminus ofa hybridized nucleic acid probe. In a preferred one-pot embodiment, thedepolymerizing enzyme is thermostable and more preferably, the treatedreaction mixture also contains (i) adenosine 5′ diphosphate, (ii)pyrophosphate, and (iii) a thermostable nucleoside diphosphate kinase(NDPK).

The treated sample is maintained under depolymerizing conditions at atemperature of about 40° C. to about 90° C., more preferably at atemperature of about 20° C. to about 90° C., and most preferably at atemperature of about 25° C. to about 80° C., for a time periodsufficient to permit the depolymerizing enzyme to depolymerize thehybridized nucleic acid probe and release an identifier nucleotide as anucleoside triphosphate. In preferred one-pot reactions, the time periodis also sufficient to permit NDPK enzyme to transfer a phosphate fromthe released nucleoside triphosphate to added ADP, thereby forming ATP.The presence or absence of a nucleic acid target sequence is determinedfrom the analytical output obtained using ATP. In a preferred method ofthe invention, analytical output is obtained by luminescencespectrometry.

In another aspect of the thermostable enzyme one-pot method fordetermining the presence or absence of a predetermined nucleic acidtarget sequence in a nucleic acid sample, the treated sample is formedby the following further steps. A hybridization composition is formed byadmixing the sample to be assayed with one or more nucleic acid probesunder hybridizing conditions. The 3′-terminal region of the nucleic acidprobe (i) hybridizes with partial or total complementarity to a nucleicacid target sequence when that sequence is present in the sample, and(ii) includes an identifier nucleotide. A treated sample is formed bymaintaining the hybridization composition under hybridizing conditionsfor a time period sufficient for the predetermined nucleic acid targetsequence to hybridize with the nucleic acid probe.

Preferably, the depolymerizing enzyme is from a group of thermophilicDNA polymerases comprising Tne triple mutant DNA polymerase, Tne DNApolymerase, Taq DNA polymerase, Ath DNA polymerase, Tvu DNA polymerase,Bst DNA polymerase, and Tth DNA polymerase. In another aspect of themethod, the NDPK is that encoded for by the thermophilic bacteriaPyrococcus furiosis (Pfu).

Another embodiment of the invention contemplates a method for enhancingthe discrimination of analytical output between a target/probe hybridwith a matched base at an interrogation position and a substantiallyidentical target/probe hybrid with a mismatched base at the sameinterrogation position. This embodiment is useful for the determinationof the presence or absence of a predetermined nucleic acid in a targetnucleic acid sequence in a nucleic acid sample, and comprises thefollowing steps.

A plurality of separate treated samples is provided. Each treated samplecontains a nucleic acid target sequence that may contain thepredetermined nucleic acid. Each nucleic acid target sequence ishybridized with a nucleic acid probe.

A first probe of a first treated sample comprises a 3′-terminal regionsequence that is complementary to the nucleic acid target sequence. Thefirst probe includes an identifier nucleotide. This identifiernucleotide is complementary to the first-named target nucleic acid. Thefirst probe also contains a second sequence otherwise complementary tothe nucleic acid target sequence except for a second predeterminednucleotide that is not complementary to the target sequence and islocated about 2 to about 10 nucleotides upstream from the 3′-terminalnucleotide of the first probe. This second predetermined nucleotidemismatched base acts to further destabilize the hybridized probe andenhances discrimination as discussed above.

A second probe of a second treated sample contains a 3′-terminal regionsequence that is complementary to the same nucleic acid target sequenceas the first probe, except for an identifier nucleotide in the3′-terminal region that is not complementary to the first predeterminednucleic acid of the first-named target nucleic acid. The second probealso contains a second sequence otherwise complementary to the targetsequence except for a second predetermined nucleotide (the same secondpredetermined nucleotide as the first probe) that is not complementaryto the target sequence and exists about 2 to about 10 nucleotidesupstream from the 3′-terminal nucleotide of said probe.

Each treated sample is admixed with a depolymerizing amount of an enzymewhose activity is to release one or more nucleotides from the3′-terminus of a hybridized nucleic acid probe to form a treatedreaction mixture. The treated reaction mixtures are maintained for atime period sufficient to permit the enzyme to depolymerize a hybridizednucleic acid probe and release an identifier nucleotide therefrom.

The samples are analyzed for the presence or absence of releasedidentifier nucleotide to obtain an analytical output. Analysis mayinclude conversion of released identifier nucleotide to ATP by NDPK inthe presence of ADP, followed by analysis of the amount of ATP present.

The ratio of the analytical output from the sample containing the firstprobe relative to that of the sample containing the second probe isenhanced compared to the ratio of the analytical output from a similarset of treated samples (samples 3 and 4) that contain the same targetand probes as do samples 1 and 2 respectively, but whose probes do notcontain the second predetermined nucleic acid that is not complementary[destabilizing mismatched base(s)] when hybridized with the target. Thissecond predetermined nucleic acid in the first and second probes islocated at about 2 to about 10 nucleotides upstream from the 3′ terminalnucleotide of the probe, preferably 3 to 6 nucleotides upstream.Therefore, the third probe is identical to the first probe except thatthe third probe does not contain a nucleotide, located at about 2 toabout 10 nucleotides upstream from the 3′ terminal nucleotide, that is amismatch when the probe is hybridized to the target sequence. Likewise,the fourth probe is identical to the second probe except that the fourthprobe does not contain a nucleotide, located at about 2 to about 10nucleotides upstream from the 3′ terminal nucleotide that is a mismatchwhen the probe is hybridized to the target sequence. Instead, the thirdand fourth probes have a complementary base at the same position as thesecond predetermined nucleic acid that is not complementary in the firstand second probes. The nucleic acid target sequence is substantially thesame for all four probes.

A still further method of the invention contemplates determining whetherthe presence or absence of a nucleic acid target sequence in a nucleicacid sample results from a locus that is homozygous or heterozygous forthe two alleles at the locus. This method is comprised of the followingsteps. A plurality of separate treated samples is provided. Each samplemay contain a nucleic acid target sequence hybridized with a nucleicacid probe. The nucleic acid target sequence consists of either a firstallele, a second allele, or a mixture of first and second alleles of thenucleic acid target. The alleles differ in sequence at an interrogationposition. The nucleic acid probe contains an identifier nucleotide inthe 3′-terminal region that is aligned at an interrogation nucleotideposition of the target sequence when the probe and target arehybridized.

A treated reaction mixture is formed by admixing each treated samplewith a depolymerizing amount of an enzyme whose activity is to releaseone or more nucleotides from the 3′-terminus of a hybridized nucleicacid probe. The treated reaction mixture is maintained underdepolymerizing conditions for a time period sufficient to permit theenzyme to depolymerize the hybridized nucleic acid probe and release anidentifier nucleotide. The samples are analyzed for the presence orabsence of released identifier nucleotides to obtain an analyticaloutput. The analytical output is quantifiable and thus determineswhether the sample is homozygous or heterozygous when compared to theanalytical output of appropriate controls.

A multiplexed version of this embodiment is also contemplated, whereinprobes for two or more alleles are provided, each distinguishable, butpreferably having the same lengths. Then, after hybridization,depolymerization, and analysis according to the invention, the relativeanalytical output for the various distinguishable identifier nucleotidesor remaining probes will show whether the sample is homozygous orheterozygous and for which alleles. Another multiplexed version of thisembodiment is contemplated, wherein probes for alleles at a plurality ofloci are provided. Preferably, the different loci have substantiallydifferent target sequences. Probes for the various alleles at each locusare preferably of the same length. Each of the probes should bedistinguishable either by analysis of the released identifier nucleotideor the remaining probe after depolymerization.

A still further embodiment of the invention contemplates determining thepresence or absence of a nucleic acid target sequence in a nucleic acidsample with a probe that is hybridized to the target and then modifiedto be able to form a hairpin structure. This embodiment comprises thefollowing steps.

A treated sample is provided that contains a nucleic acid sample thatmay include a nucleic acid target sequence having an interrogationposition hybridized with a nucleic acid probe. The probe is comprised ofat least two sections. The first section contains the probe 3′-terminalabout 10 to about 30 nucleotides. These nucleotides are complementary tothe target strand sequence at positions beginning about 1 to about 30nucleotides downstream of the interrogation position. The second sectionof the probe is located at the 5′-terminal region of the probe andcontains about 10 to about 20 nucleotides of the target sequence. Thissequence spans the region in the target from the nucleotide at or justupstream (5′) of the interrogation position, to the nucleotide justupstream to where the 3′-terminal nucleotide of the probe anneals to thetarget. An optional third section of the probe, from zero to about 50,and preferably about zero to about 20 nucleotides in length andcomprising a sequence that does not hybridize with either the first orsecond section, is located between the first and second sections of theprobe.

The probe of the treated sample is extended in a template-dependentmanner, as by admixture with dNTPs and a template-dependent polymerase,at least through the interrogation position, thereby forming an extendedprobe/target hybrid. In a preferred embodiment, the length of the probeextension is limited by omission from the extension reaction of a DNTPcomplementary to a nucleotide of the target sequence that is presentupstream of the interrogation position and absent between the nucleotidecomplementary to the 3′-end of the interrogation position.

The extended probe/target hybrid is separated from any unreacted dNTPs.The extended probe/target hybrid is denatured to separate the strands.The extended probe strand is permitted to form a hairpin structure.

A treated reaction mixture is formed by admixing the hairpinstructure-containing composition with a depolymerizing amount of anenzyme whose activity is to release one or more nucleotides from the3′-terminus of an extended probe hairpin structure. The reaction mixtureis maintained under depolymerizing conditions for a time periodsufficient for the depolymerizing enzyme to release 3′-terminusnucleotides, and then analyzed for the presence of released identifiernucleotides. The analytical output indicates the presence or absence ofthe nucleic acid target sequence.

A still further embodiment of the invention, termed REAPER™, alsoutilizes hairpin structures. This method contemplates determining thepresence or absence of a nucleic acid target sequence, or a specificbase within the target sequence, in a nucleic acid sample, and comprisesthe following steps. A treated sample is provided that contains anucleic acid sample that may include a nucleic acid target sequencehybridized with a first nucleic acid probe strand.

The hybrid is termed the first hybrid. The first probe is comprised ofat least two sections. The first section contains the probe 3′-terminalabout 10 to about 30 nucleotides that are complementary to the targetnucleic acid sequence at a position beginning about 5 to about 30nucleotides downstream of the target interrogation position. The secondsection of the first probe contains about 5 to about 30 nucleotides thatare a repeat of the target sequence from the interrogation position toabout 10 to about 30 nucleotides downstream of the interrogationposition, and does not hybridize to the first section of the probe. Anoptional third section of the probe, located between the first andsecond sections of the probe, is zero to about 50, preferably up toabout 20, nucleotides in length and comprises a sequence that does nothybridize to either the first or second section.

The first hybrid in the treated sample is extended at the 3′-end of thefirst probe, thereby extending the first probe past the interrogationposition and forming an extended first hybrid whose sequence includes aninterrogation position. The extended first hybrid is comprised of theoriginal target nucleic acid and extended first probe. The extendedfirst hybrid is then denatured in an aqueous composition to separate thetwo nucleic acid strands of the hybridized duplex and form an aqueoussolution containing a separated target nucleic acid and a separatedextended first probe.

A second probe, that is about 10 to about 2000, preferably about 10 toabout 200, most preferabley about 10 to about 30 nucleotides in lengthand is complementary to the extended first probe at a position beginningabout 5 to about 2000, preferably about 5 to about 200, nucleotidesdownstream of the interrogation position in extended first probe, isannealed to the extended first probe, thereby forming the second hybrid.The second hybrid is extended at the 3′-end of the second probe untilthat extension reaches the 5′-end of the extended first probe, therebyforming a second extended hybrid whose 3′-region includes an identifiernucleotide. In preferred embodiments the extending polymerase for bothextensions does not add a nucleotide to the 3′ end that does not have acorresponding complementary nucleotide in the template.

An aqueous composition of the extended second hybrid is denatured toseparate the two nucleic acid strands. The aqueous composition so formedis cooled to form a “hairpin structure” from the separated extendedsecond probe when the target sequence is present in the original nucleicacid sample.

A treated reaction mixture is formed by admixing the hairpinstructure-containing composition with a depolymerizing amount of anenzyme whose activity is to release one or more nucleotides from the3′-terminus of a nucleic acid hybrid. The reaction mixture is maintainedunder depolymerizing conditions for a time period sufficient to release3′-terminal region identifier nucleotides, and then analyzed for thepresence of released identifier nucleotides. The analytical outputindicates the presence or absence of the nucleic acid target sequence.

A further method of the invention contemplates a method for determiningthe presence or absence of a restriction endonuclease recognitionsequence in a nucleic acid sample that comprises the following steps. Atreated sample is provided that may contain a hybridized nucleic acidtarget that has a cleaved restriction endonuclease recognition sequencethat includes an identifier nucleotide in the restriction endonucleaserecognition sequence. The treated sample is admixed with adepolymerizing amount of an enzyme whose activity is to release one ormore nucleotides from the 3′-terminus of a restriction endonucleaserecognition sequence to form a treated reaction mixture. The treatedreaction mixture is maintained under depolymerizing conditions for atime period sufficient to permit the enzyme to depolymerize hybridizednucleic acid and release identifier nucleotide therefrom. Finally, thetreated reaction mixture is analyzed for the presence of releasedidentifier nucleotides to obtain an analytical output, the analyticaloutput indicating the presence or absence of the restrictionendonuclease recognition sequence in the nucleic acid target.

In a preferred embodiment, the process includes the further steps offorming a treated sample by providing an endonuclease cleavage reactionsolution comprising a nucleic acid sample and a restriction endonucleaseenzyme specific for the restriction endonuclease recognition sequenceand maintaining the endonuclease cleavage reaction solution for a timeperiod sufficient for the restriction endonuclease enzyme to cleave therestriction endonuclease recognition sequence to form a treated sample.

In a further preferred embodiment, the process includes the further stepof amplifying a nucleic acid target sequence in a nucleic acid sampleprior to providing the restriction endonuclease recognition sequence.

In a still further preferred embodiment, the nucleic acid targetsequence of the process is amplified by the following further steps.

A crude nucleic acid sample is admixed with PCR amplification primersthat are complementary to regions upstream and downstream of the nucleicacid target sequence and a template-dependent polymerase to form anamplification sample mixture wherein either the nucleic acid targetsequence or the PCR amplification primers includes a restrictionendonuclease recognition sequence. The amplification sample mixture ismaintained under denaturing conditions for a time period sufficient todenature the nucleic acid target sequence to form a denaturedamplification reaction mixture. The denatured amplification mixture isannealed under hybridization conditions for a time period sufficient forPCR amplification primers to anneal to the nucleic acid target sequenceto form an amplification reaction mixture. Finally, the amplificationreaction mixture is maintained for a time period sufficient to permitthe template-dependent polymerase to extend the nucleic acid from thePCR primers to form an amplified nucleic acid sample. The amplificationcycle is repeated as usual for PCR to obtain a PCR amplification productthat has restriction endonuclease recognition sites in it. This PCRamplification product is purified and reacted with a depolymerizingenzyme as described above.

Another embodiment of the invention contemplates a method fordetermining the loss of heterozygosity (LOH) of a locus of an allelethat comprises the following steps.

A plurality of separate treated samples is provided, each samplecontaining a nucleic acid target sequence hybridized with a nucleic acidprobe. The nucleic acid target sequence is that of a first allele or amixture of the first allele and a second allele of the nucleic acidtarget, wherein the alleles differ in sequence. The nucleic acid probecontains a 3′-terminal region that hybridizes to a target sequence whenthe probe and target are hybridized.

Each treated sample is admixed with a depolymerizing amount of an enzymewhose activity is to release one or more nucleotides from the3′-terminus of a hybridized nucleic acid probe to form a treatedreaction mixture. The treated reaction mixture is maintained underdepolymerizing conditions for a time period sufficient to depolymerizehybridized nucleic acid probe and release identifier nucleotides. Thesamples are then analyzed for the quantity of released identifiernucleotides to obtain an analytical output, the analytical outputindicating whether the nucleic acid target sequence in a nucleic acidsample has lost heterozygosity at the locus of the allele.

In preferred LOH embodiments, the analytical output is obtained byluminescence spectroscopy, absorbance spectrometry, mass spectrometry orfluorescence spectroscopy. In another preferred embodiment, the releasedidentifier nucleotide includes a fluorescent label. The identifiernucleotide is optionally fluorescently labeled after release from thehybrid. It is contemplated that in the above analytical methods, eitherthe released identifier nucleotide or the remainder of the probe can beevaluated to determine whether identifier nucleotide had been released,as described herein.

In another preferred LOH embodiment, the enzyme whose activity is torelease nucleotides is a template-dependent polymerase that, in thepresence of pyrophosphate ions, depolymerized hybridized nucleic acidswhose bases in the 3′-terminal region are completely complementary tobases of the nucleic acid target.

In one aspect of the LOH embodiment, the quantity of the releasedidentifier nucleotides for the first allele is substantially less thanthe quantity of the released identifier nucleotide for the first alleleof a known heterozygous control sample, and the quantity of the releasedidentifier nucleotides for the second allele is substantially similar tothat of the released identifier nucleotide for the second allele of aknown heterozygous control sample, indicating a loss of heterozygosityat the locus of the first allele.

In another aspect of the LOH embodiment, the quantity of the releasedidentifier nucleotides for the second allele is substantially less thanthe quantity of the released identifier nucleotides for the secondalleles of a known heterozygous control sample, and the quantity of thereleased identifier nucleotides for the first allele is substantiallysimilar to that of the released identifier nucleotide for the firstallele of a known heterozygous control sample, indicating a loss ofheterozygosity at the locus of the second allele. The known heteozygouscontrol has analytical output for the treated samples indicating allelesone and two are present in the sample at about a 1:1 ratio. A samplewith loss of heterozygosity has an analytical output for the treatedsamples indicating alleles one and two are present in the sample at a1:0 or 0:1 ratio respectively when compared to the analytical output ofa known heterozygous control sample.

A still further preferred embodiment of the invention contemplates amethod for determining the presence of trisomy of an allele thatcomprises the following steps.

A plurality of separate treated samples is provided, wherein each samplecontains a nucleic acid target sequence hybridized with a nucleic acidprobe. The nucleic acid target sequence is that of a first allele, asecond allele or a mixture of the first and second alleles of thenucleic acid target. The alleles differ in sequence at an interrogationposition. The nucleic acid probe contains a 3′-terminal region thathybridizes to a region of the nucleic acid target sequence containingthe interrogation nucleotide position when the probe and target arehybridized. The nucleic acid probe also contains an identifiernucleotide.

Each treated sample is admixed with a depolymerizing amount of an enzymewhose activity, under depolymerizing conditions, is to release one ormore nucleotides from the 3′-terminus of a hybridized nucleic acid probeto form a treated reaction mixture. The treated reaction mixture ismaintained for a time period sufficient to depolymerize hybridizednucleic acid probe and release identifier nucleotides. The samples areanalyzed for released identifier nucleotides to obtain an analyticaloutput, the magnitude of the analytical output relative to an analyticaloutput of an appropriate control sample indicating whether a trisomy ispresent in the nucleic acid target sequence.

For trisomy analysis, preferably the analytical output is obtained byluminescence spectroscopy, absorbance spectrometry, fluorescencespectroscopy, or mass spectrometry. the released identifier nucleotidepreferably includes a fluorescent label. The identifier nucleotide isoptionally fluorescently labeled after release from the hybrid.

In a preferred embodiment for trisomy analysis, the enzyme whoseactivity is to release nucleotides is a template-dependent polymerase,that, in the presence of pyrophosphate ions, depolymerizes hybridizednucleic acids whose bases in the 3′ terminal region are completelycomplementary to bases of said nucleic acid target.

In one embodiment, the quantity of the released identifier nucleotidefor the first allele is substantially greater than the quantity of thereleased identifier nucleotide of a control sample homozygous for thefirst allele, indicating that the nucleic acid target sequence has atrisomy. Preferably, the quantity of released identifier is expressed asa ratio. For example, a normal heterozygote has about a 1:1 ratio of theanalytical output for the two alleles. If the trisomy is homozygous foreither allele, the ratio is about three times the value for that allelein a normal heterozygote that has none of the other allele. If thetrisomy is heterozygous, then the ratio is about 2:1 of one allele tothe other when compared to the analytical output of a controlheterozygote.

An embodiment of the invention contemplates a process to determine thepresence or absence of a predetermined single-stranded nucleic acidtarget sequence. Such a process comprises the following steps.

A depolymerization reaction mixture is provided that comprises a pair offirst and second nucleic acid probes and a hybrid between a third probeand the nucleic acid target sequence. The first and second nucleic acidprobes are complementary and form 3′-overhangs on both ends of theduplex formed when each of the pair of complementary nucleic acid probesis hybridized with the other. The first of those probes is complementaryto the nucleic acid target sequence, whereas the second has the sequenceof the nucleic acid target. A hybrid between a third probe and thenucleic acid target sequence is present in the depolymerization reactionmixture when the nucleic acid target sequence is present in the nucleicacid sample. Each of the first and third probes has an identifiernucleotide in its 3′-terminal region. The reaction mixture furthercomprises a depolymerizing amount of an enzyme whose activity is torelease nucleotides from the 3′-terminus of a hybridized nucleic acid.

The reaction mixture is maintained under depolymerization conditions fora time period sufficient to permit the enzyme to depolymerize the3′-terminal region of the hybridized third probe to release identifiernucleotides and form a first treated reaction mixture.

The products of the first treated reaction mixture are denatured to forma denatured treated reaction mixture.

The denatured treated reaction mixture is maintained under hybridizingconditions for a time period sufficient to form a seconddepolymerization reaction mixture. That second depolymerization reactionmixture comprises two components. The first is a hybrid formed betweenthe first probe and the nucleic acid target sequence, when the nucleicacid target sequence is present in the nucleic acid sample. The secondcomponent is a hybrid formed between the 3′-terminal-depolymerized thirdprobe and the second nucleic acid probe. One end of that second hybridhas a blunt end or a 5′-overhang, as well as an identifier nucleotide inthe 3′-terminal region.

The first and second hybrid components of the second reaction mixtureare depolymerized to release identifier nucleotide from the 3′-terminalregions of the hybrids to form a second treated reaction mixture. Thesecond treated reaction mixture is analyzed for the presence of releasedidentifier nucleotide to obtain an analytical output, the analyticaloutput indicating the presence or absence of said nucleic acid targetsequence.

In preferred practice, the first and third probes are the same. Inaddition, the denaturation, annealing and depolymerization steps arepreferably repeated to further increase the number of nucleic acidhybrids from which identifier nucleotides are released prior to analysisof the amplification reaction mixture to detect released identifiernucleotide. Most preferably, the depolymerizing enzyme is thermostable.

A related embodiment of the invention contemplates a process todetermine the presence or absence of a predetermined double-strandednucleic acid target sequence. The process comprises the following steps.

A first reaction mixture comprises first and second nucleic acid probes,third and fourth nucleic acid probes, and a depolymerizing enzyme. Thefirst and second complementary nucleic acid probes form 3′-overhangs onboth ends of the duplex formed when each of the complementary nucleicacid probes is hybridized with the other. Each of those probes iscomplementary to one or the other strand of the nucleic acid targetsequence and has an identifier nucleotide in its 3′-terminal region.Hybrids between a third and fourth probe and each of the two strands ofthe nucleic acid target sequence are present when the nucleic acidtarget sequence is present in the nucleic acid sample. The third andfourth probes each have an identifier nucleotide in its 3′-terminalregion. Also present is a depolymerizing amount of a depolymerizingenzyme whose activity is to release nucleotides from the 3′-terminus ofa hybridized nucleic acid.

The first reaction mixture is maintained for a time period sufficient topermit the enzyme to depolymerize hybridized nucleic acid to releaseidentifier nucleotide from the 3′-terminal region of the hybridizedthird and fourth probes and form a treated first reaction mixture. Theproducts of the treated first reaction mixture are denatured to form adenatured treated reaction mixture.

The denatured treated reaction mixture is maintained under hybridizingconditions for a time period sufficient to form a second reactionmixture. That second reaction mixture comprises two components. A firstcomponent is comprised of hybrids that lack a 3′-overhang between eachof the strands of the target nucleic acid and each of the first andsecond probes, when the nucleic acid target sequence is present in thenucleic acid sample. A second component is comprised of hybrids betweeneach of the first and second probes and 3′-terminal region-depolymerizedthird and fourth probes. Each of the hybrids of each of the componentscontains one end that is blunt or has a 5′-overhang, as well as anidentifier nucleotide in the 3′-terminal region.

The hybrids of the first and second components above are depolymerizedto release identifier nucleotide from the 3′-terminus of the hybridizedprobes to form a second treated reaction mixture. The second treatedreaction mixture is analyzed for the presence of released identifiernucleotide to obtain an analytical output, the analytical outputindicating the presence or absence of said nucleic acid target sequence.

In preferred practice, the first and third probes are the same. Inaddition, the denaturation, annealing and depolymerization steps arepreferably repeated to further increase the number of nucleic acidhybrids from which identifier nucleotides are released prior to analysisof the amplification reaction mixture to detect released identifiernucleotide.

A further embodiment of the invention contemplates an isolated andpurified nucleotide diphosphate kinase (NDPK) enzyme that exhibitshigher NDPK activity at a temperature of about 50 to about 90 degrees C.relative to the NDPK activity at 37 degrees C. Such an NDPK is from Pfuand comprises the amino acid sequence of SEQ ID NO:90. Preferably, theisolated and purified NDPK enzyme has a DNA sequence shown in SEQ IDNO:91.

In a contemplated amplification and interrogation process of theinvention, the presence or absence of a predetermined nucleic acidtarget sequence is determined, comprising the following steps. Aligation reaction solution is provided, comprising a ligating amount ofa ligase, a nucleic acid sample and an open circle probe. The nucleicacid sample may contain the predetermined nucleic acid target sequence.The nucleic acid target sequence has a 3′-portion and a 5′-portion. Theopen circle probe has an open circle probe 3′-terminal region, a linkerregion, and an open circle probe 5′-terminal region. The open circleprobe further comprises a detection primer target and an amplificationprimer target. The amplification primer target is downstream of thedetection primer target.

Upon hybridization between the open circle probe and the predeterminednucleic acid target sequence, the open circle probe 3′-terminal regionis complementary to a sequence of the 3′ portion of the nucleic acidtarget sequence. Similarly, the open circle probe 5′-terminal region iscomplementary to a sequence of the 5′ portion of the nucleic acid targetsequence.

The ligation reaction solution optionally further comprises apolymerizing amount of a DNA polymerase and deoxynucleosidetriphosphates. Preferably, this is when the hybridized open circle probe3′-terminus is not adjacent and ligatable to the hybridized open circleprobe 5′-terminus and a gap is present between those termini.

The ligation reaction solution is maintained for a time periodsufficient to permit the filling-in of the gap when DNA polymerase ispresent, and also for a time period sufficient for ligation of thetermini of the open circle probe to form a closed circular probe, andthus a treated ligation reaction solution.

The closed circular probe is admixed with an amplification primer, whichhybridizes with the amplification primer target, nucleosidetriphosphates, and a polymerizing amount of a DNA polymerase to form areplication reaction mixture. The replication reaction mixture ismaintained for a time period sufficient to permit the extension of anucleic acid strand from the amplification primer. The extension productnucleic acid strand comprises an interrogation target to form a treatedreplication mixture.

An interrogation probe is admixed with the treated replication mixture.The interrogation probe is complementary to the interrogation target,and comprises an identifier nucleotide in its 3′-terminal region. Thetreated replication mixture is denatured before, during or afteraddition of the probe to form a denatured mixture. Preferably, thetreated replication mixture is denatured after addition of the probe.The denatured mixture is annealed to permit the formation of a hybridbetween the interrogation probe and the interrogation target, whenpresent, thus forming an interrogation solution. A depolymerizing amountof an enzyme, whose activity is to release one or more nucleotides fromthe 3′-terminus of a hybridized nucleic acid probe, is admixed with theinterrogation solution, thus forming a depolymerization reactionmixture. The depolymerization and analysis for released identifiernucleotide is as described above.

Preferably, the depolymerizing enzyme is thermostable. In a preferredembodiment, the free nucleotide triphosphates are separated from thetreated replication mixture prior to the depolymerization step.

Optionally, in the above process wherein there is a gap present betweenthe termini of the hybridized open circle probe, the portion of thepredetermined nucleic acid target sequence between the 3′- and5′-termini of the hybridized open circle probe that is opposite the gapcontains three or fewer nucleotides and only nucleoside triphosphatescomplementary to the three or fewer nucleotides are present in theligation reaction solution. Preferably, a polymerizing amount of a DNApolymerase and nucleoside triphosphates are present in the ligationreaction solution. Optionally, the open circle probe comprises aplurality of detection primer targets. Preferably, in the above process,the presence or absence of a plurality of predetermined nucleic acidtargets is determined using a plurality of detection probes comprisingdifferent identifier nucleotides. Analysis of the released identifiernucleotide is preferably done according to methods disclosed elsewhereherein.

Another embodiment of the invention contemplates an amplification andinterrogation process to determine the presence or absence of apredetermined nucleic acid target sequence having a 3′-portion and a5′-portion comprising the following steps.

A ligation reaction solution is provided, comprising (i) a ligatingamount of a ligase, (ii) a nucleic acid sample that may contain apredetermined nucleic acid target sequence wherein the nucleic acidtarget sequence has a 3′-portion and a 5′-portion, (iii) a pair ofligation probes, the ligation probe further including a detection primertarget and an amplification primer target, the amplification primertarget being downstream of the detection primer target, wherein uponhybridization between the open circle probe and the nucleic acid targetsequence, the open circle probe 3′-terminal region is complementary to asequence of the 3′-portion of the predetermined nucleic acid targetsequence, and the open circle probe 5′-terminal region is complementaryto a sequence of the 5′-portion of said predetermined nucleic acidtarget sequence, and (iv) optionally further comprising a polymerizingamount of a DNA polymerase and deoxynucleoside triphosphates when thehybridized open circle probe 3′-terminus is not adjacent and ligatableto the hybridized open circle probe 5′-terminus and a gap is presentbetween those termini.

The ligation reaction solution is maintained for a time periodsufficient to permit filling-in of the gap, when present, and ligationof the termini of the open circle probe to form a closed circular probeand a treated ligation reaction solution.

The closed circular probe is admixed with an amplification primer thathybridizes with the amplification primer target, nucleosidetriphosphates, and a polymerizing amount of a DNA polymerase to form areplication reaction mixture.

The replication reaction mixture is maintained for a time periodsufficient to permit extension of a nucleic acid strand from theamplification primer, wherein the extension product nucleic acid strandcomprises a interrogation target to form a treated replication mixture.

An interrogation probe is admixed with the treated replication mixture,wherein the interrogation probe is complementary to the interrogationtarget and comprises an identifier nucleotide in the 3′-terminal region.

The treated replication mixture is denatured to form a denaturedmixture.

The denatured mixture is annealed to form a hybrid between theinterrogation probe and the interrogation target when present to form aninterrogation solution.

A depolymerizing amount of an enzyme whose activity is to release one ormore nucleotides from the 3′-terminus of a hybridized nucleic acid probeis admixed with the interrogation solution to form a depolymerizationreaction mixture.

The depolymerization reaction mixture is maintained under depolymerizingconditions for a time period sufficient to permit the enzyme todepolymerize hybridized nucleic acid and release identifier nucleotidetherefrom. The presence of released identifier nucleotide is analyzed toobtain an analytical output, the analytical output indicating thepresence or absence of the predetermined nucleic acid target sequence.

Preferably, the depolymerizing enzyme is thermostable. In one embodimentof the process, free nucleotide triphosphates are separated from thetreated replication mixture prior to step admixing a depolymerizingamount of an enzyme whose activity is to release one or more nucleotidesfrom the 3′-terminus of a hybridized nucleic acid probe with theinterrogation solution to form a depolymerization reaction mixture. Inanother embodiment of the process, there is a gap present between thetermini of the hybridized open circle probe, the portion of thepredetermined nucleic acid target sequence between the 3′- and5′-termini of the hybridized open circle probe that is opposite the gapcontains three or fewer nucleotides and only nucleoside triphosphatescomplementary to the three or fewer nucleotides are present in theligation reaction solution. Preferably, a polymerizing amount of a DNApolymerase and nucleoside triphosphates are present in the ligationreaction solution.

Optionally, the open circle probe comprises a plurality of detectionprimer targets. Preferably, the presence or absence of a plurality ofpredetermined nucleic acid targets is determined using a plurality ofdetection probes comprising different identifier nucleotides.

Optionally, analysis of the released identifier nucleotides is by massspectrometry.

The present invention has many benefits and advantages, several of whichare listed below.

One benefit of the invention is that, in some embodiments, nucleic acidhybrids can be detected with very high levels of sensitivity without theneed for radiochemicals or electrophoresis.

An advantage of the invention is that the presence or absence of one ormore target nucleic acid(s) can be detected reliably, reproducibly, andwith great sensitivity.

A further benefit of the invention is that quantitative information canbe obtained about the amount of a target nucleic acid sequence in asample.

A further advantage of the invention is that very slight differences innucleic acid sequence are detectable, including single nucleotidepolymorphisms (SNPs).

Yet another benefit of the invention is that the presence or absence ofa number of target nucleic acid sequences can be determined in the sameassay.

Yet another advantage of the invention is that the presence or absenceof a target nucleic acid can be determined with a small number ofreagents and manipulations.

Another benefit of the invention is that the processes lend themselvesto automation.

Still another benefit of the invention is its flexibility of use in manydifferent types of applications and assays including, but not limitedto, detection of mutations, translocations, and SNPs in nucleic acid(including those associated with genetic disease), determination ofviral load, species identification, sample contamination, and analysisof forensic samples.

Still further benefits and advantages of the invention will becomeapparent from the specification and claims that follow.

BRIEF DESCRIPTION OF THE DRAWING

In the drawings forming a portion of this disclosure,

FIG. 1 illustrates the annealing of the 10865 oligonucleotide (SEQ IDNO:196) to 10870 wild type (SEQ ID NO:194) and 10994 mutant (SEQ IDNO:195) oligonucleotides utilized in rolling circle amplification asFIG. 1A and FIG. 1B, respectively. Also shown are the annealing(hybridization) of oligonucleotide 10866 to oligonucleotide 10865, aswell as the hybridization of oligonucleotide probe 10869 (SEQ ID NO:198)to oligonucleotide 10870 and of oligonucleotide probe 10989 (SEQ IDNO:199) to oligonucleotide 10994 as representations of the binding ofthose probes to the respective amplified sequences. Arcuate lines inoligonucleotide 10865 are used to help illustrate the shape thatoligonucleotide 10865 can assume when hybridized with either ofoligonucleotides 10870 or 10994.

FIG. 2. illustrates the Reaper™ assay as illustrated in Example 89. FIG.2A illustrates the first hybrid formed by the annealing of nucleic acidtarget SEQ ID NO: 286 (286) to first probe SEQ ID NO: 287 (287). Anarrow points to an interrogation position in 286.

FIG. 2B illustrates the first extended hybrid formed by the annealing of286 to the extended 287. Extended 287 is first extended probe SEQ ID NO:288 (288).

FIG. 2C illustrates the second hybrid formed by annealing of 288 fromthe denatured nucleic acid molecule shown in FIG. 2B to the second probedenoted SEQ ID NO: 289 (289). An arrow points to the interrogationposition in 288.

FIG. 2D illustrates the extended second hybrid formed by the annealingof 288 and the extended 289 strand denoted SEQ ID NO: 290 (290).

FIG. 2E illustrates the 290 strand denatured from FIG. 2D and forming ahairpin structure. An arrow points to the interrogation position at the3′-terminus of the hybrid.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below. “Nucleoside”, as used herein, refers to a compoundconsisting of a purine [guanine (G) or adenine (A)] or pyrimidine[thymine (T), uridine (U) or cytidine (C)] base covalently linked to apentose, whereas “nucleotide” refers to a nucleoside phosphorylated atone of its pentose hydroxyl groups. “XTP”, “XDP” and “XMP” are genericdesignations for ribonucleotides and deoxyribonucleotides, wherein the“TP” stands for triphosphate, “DP” stands for diphosphate, and “MP”stands for monophosphate, in conformity with standard usage in the art.Subgeneric designations for ribonucleotides are “NMP”, “NDP” or “NTP”,and subgeneric designations for deoxyribonucleotides are “dNMP”, “DNDP”or “dNTP”. Also included as “nucleoside”, as used herein, are materialsthat are commonly used as substitutes for the nucleosides above such asmodified forms of these bases (e.g. methyl guanine) or syntheticmaterials well known in such uses in the art, such as inosine.

A “nucleic acid,” as used herein, is a covalently linked sequence ofnucleotides in which the 3′ position of the pentose of one nucleotide isjoined by a phosphodiester group to the 5′ position of the pentose ofthe next, and in which the nucleotide residues (bases) are linked inspecific sequence; i.e., a linear order of nucleotides. A“polynucleotide,” as used herein, is a nucleic acid containing asequence that is greater than about 100 nucleotides in length. An“oligonucleotide,” as used herein, is a short polynucleotide or aportion of a polynucleotide. An oligonucleotide typically contains asequence of about two to about one hundred bases. The word “oligo” issometimes used in place of the word “oligonucleotide”.

A base “position” as used herein refers to the location of a given baseor nucleotide residue within a nucleic acid.

A “nucleic acid of interest,” as used herein, is any particular nucleicacid one desires to study in a sample.

The term “isolated” when used in relation to a nucleic acid or protein,refers to a nucleic acid sequence or protein that is identified andseparated from at least one contaminant (nucleic acid or protein,respectively) with which it is ordinarily associated in its naturalsource. Isolated nucleic acid or protein is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids or proteins are found in the state they existin nature.

As used herein, the term “purified” or “to purify” means the result ofany process which removes some contaminants from the component ofinterest, such as a protein or nucleic acid. The percent of a purifiedcomponent is thereby increased in the sample.

The term “wild-type,” as used herein, refers to a gene or gene productthat has the characteristics of that gene or gene product that is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” as used herein, refers to a gene or gene productthat displays modifications in sequence and/or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product.

Nucleic acids are known to contain different types of mutations. As usedherein, a “point” mutation refers to an alteration in the sequence of anucleotide at a single base position. A “lesion”, as used herein, refersto site within a nucleic acid where one or more bases are mutated bydeletion or insertion, so that the nucleic acid sequence differs fromthe wild-type sequence.

A “single nucleotide polymorphism” or SNP, as used herein, is avariation from the most ifrequently occurring base at a particularnucleic viacid position.

Homologous genes or alleles from different species are also known tovary in sequence. Regions of homologous genes or alleles from differentspecies can be essentially identical in sequence. Such regions arereferred to herein as “regions of identity.” It is contemplated hereinthat a “region of substantial identity” can contain some “mismatches,”where bases at the same position in the region of identity aredifferent. This base position is referred to herein as “mismatchposition.”

DNA molecules are said to have a “5′-terminus” (5′ end) and a“3′-terminus” (3′ end) because nucleic acid phosphodiester linkagesoccur to the 5′ carbon and 3′ carbon of the pentose ring of thesubstituent mononucleotides. The end of a polynucleotide at which a newlinkage would be to a 5′ carbon is its 5′ terminal nucleotide. The endof a polynucleotide at which a new linkage would be to a 3′ carbon isits 3′ terminal nucleotide. A terminal nucleotide, as used herein, isthe nucleotide at the end position of the 3′- or 5′-terminus. As usedherein, a nucleic acid sequence, even if internal to a largeroligonucleotide or polynucleotide, also can be said to have 5′- and3′-ends. For example, a gene sequence located within a larger chromosomesequence can still be said to have a 5′- and 3′-end.

As used herein, the 3′-terminal region of the nucleic acid probe refersto the region of the probe including nucleotides within about 10residues from the 3′-terminal position.

In either a linear or circular DNA molecule, discrete elements arereferred to as being “upstream” or “5′” relative to an element if theyare bonded or would be bonded to the 5′-end of that element. Similarly,discrete elements are “downstream” or “3′” relative to an element ifthey are or would be bonded to the 3′-end of that element. Transcriptionproceeds in a 5′ to 3′ manner along the DNA strand. This means that RNAis made by the sequential addition of ribonucleotide-5′-triphosphates tothe 3′-terminus of the growing chain (with the elimination ofpyrophosphate).

As used herein, the term “target nucleic acid” or “nucleic acid target”refers to a particular nucleic acid sequence of interest. Thus, the“target” can exist in the presence of other nucleic acid molecules orwithin a larger nucleic acid molecule.

As used herein, the term “nucleic acid probe” refers to anoligonucleotide or polynucleotide that is capable of hybridizing toanother nucleic acid of interest. A nucleic acid probe may occurnaturally as in a purified restriction digest or be producedsynthetically, recombinantly or by PCR amplification. As used herein,the term “nucleic acid probe” refers to the oligonucleotide orpolynucleotide used in a method of the present invention. That sameoligonucleotide could also be used, for example, in a PCR method as aprimer for polymerization, but as used herein, that oligonucleotidewould then be referred to as a “primer”. Herein, oligonucleotides orpolynucleotides may contain a phosphorothioate bond.

As used herein, the terms “complementary” or “complementarity” are usedin reference to nucleic acids (i.e., a sequence of nucleotides) relatedby the well-known base-pairing rules that A pairs with T and C pairswith G. For example, the sequence 5′-A-G-T-3′, is complementary to thesequence 3′-T-C-A-5′. Complementarity can be “partial,” in which onlysome of the nucleic acid bases are matched according to the base pairingrules. On the other hand, there may be “complete” or “total”complementarity between the nucleic acid strands when all of the basesare matched according to base pairing rules. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands as known well in the art. This is of particular importance indetection methods that depend upon binding between nucleic acids, suchas those of the invention. The term “substantially complementary” refersto any probe that can hybridize to either or both strands of the targetnucleic acid sequence under conditions of low stringency as describedbelow or, preferably, in polymerase reaction buffer (Promega, M195A)heated to 95° C. and then cooled to room temperature. As used herein,when the nucleic acid probe is referred to as partially or totallycomplementary to the target nucleic acid, that refers to the 3′-terminalregion of the probe (i.e. within about 10 nucleotides of the 3′-terminalnucleotide position).

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acid strands. Hybridization and thestrength of hybridization (i.e., the strength of the association betweennucleic acid strands) is impacted by many factors well known in the artincluding the degree of complementarity between the nucleic acids,stringency of the conditions involved affected by such conditions as theconcentration of salts, the T_(m) (melting temperature) of the formedhybrid, the presence of other components (e.g., the presence or absenceof polyethylene glycol), the molarity of the hybridizing strands and theG:C content of the nucleic acid strands.

As used herein, the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “weak” or “low”stringency are often required when it is desired that nucleic acidswhich are not completely complementary to one another be hybridized orannealed together. The art knows well that numerous equivalentconditions can be employed to comprise low stringency conditions.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature”. The melting temperature is the temperature at which 50% ofa population of double-stranded nucleic acid molecules becomesdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well-known in the art. The T_(m) of a hybrid nucleicacid is often estimated using a formula adopted from hybridizationassays in 1 M salt, and commonly used for calculating T_(m) for PCRprimers: [(number of A+T)×2° C.+(number of G+C)×4° C.]. C. R. Newton etal. PCR, 2^(nd) Ed., Springer-Verlag (New York: 1997), p. 24. Thisformula was found to be inaccurate for primers longer that 20nucleotides. Id. Other more sophisticated computations exist in the artwhich take structural as well as sequence characteristics into accountfor the calculation of T_(m). A calculated T_(m) is merely an estimate;the optimum temperature is commonly determined empirically.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There can be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits a completely complementary sequence from hybridizingto a target nucleic acid is referred to using the functional term“substantially homologous.”

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous,” as usedherein, refers to a probe that can hybridize to a strand of thedouble-stranded nucleic acid sequence under conditions of lowstringency.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous,” as used herein, refers to a probe thatcan hybridize to (i.e., is the complement of) the single-strandednucleic acid template sequence under conditions of low stringency.

The term “interrogation position,” as used herein, refers to thelocation of a given base of interest within a nucleic acid probe. Forexample, in the analysis of SNPs, the “interrogation position” in theprobe is in the position that would be complementary to the singlenucleotide of the target that may be altered from wild type. Theanalytical output from a method of the invention provides informationabout a nucleic acid residue of the target nucleic acid that iscomplementary to an interrogation position of the probe. Aninterrogation position is within about ten bases of the actual3′-terminal nucleotide of the nucleic acid probe, although notnecessarily at the 3′-terminal nucleotide position. The interrogationposition of the target nucleic acid sequence is opposite theinterrogation position of the probe, when the target and probe nucleicacids are hybridized.

The term “identifier nucleotide,” as used herein, refers to a nucleotidewhose presence is to be detected in a process of the invention toidentify that a depolymerization reaction has occurred. The particularapplication of a method of the invention affects which residues areconsidered an identifier nucleotide. For a method using ATP detection(e.g. luciferase/luciferin or NADH) wherein, during analysis, allnucleotides released in the depolymerization are “converted” to ATP withan enzyme such as NDPK, all nucleotides released are identifiernucleotides. Similarly, for a method using absorbance detection thatdoes not distinguish between nucleotides, all released nucleotides areidentifier nucleotides. For a mass spectrometric detection wherein allthe released nucleotides are analyzed, all released nucleotides can beidentifier nucleotides; alternatively a particular nucleotide (e.g. anucleotide analog having a distinctive mass) can be detected. Forfluorescence detection, a fluorescently-labeled nucleotide is anidentifier nucleotide. The nucleotide may be labeled prior to or afterrelease from the nucleic acid. For radiographic detection, aradioactively-labeled nucleotide is an identifier nucleotide. In somecases, the release of identifier nucleotide is deduced by analyzing theremainder of the probe after a depolymerization step of the invention.Such analysis is generally by a determination of the size or mass of theremaining probe and can be by any of the described analytical methods(e.g. a fluorescent tag on the 5′-terminus of the probe to monitor itsmolecular weight following capillary electrophoresis).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to a class of enzymes, each of which cut double-strandedDNA. Some restriction endonucleases cut double strand DNA at or near aspecific nucleotide sequence, such as the enzyme commonly referred to asBamH I that recognizes the double strand sequence 5′GGATCC 3′. However,other representatives of such enzymes cut DNA in a non-specific mannersuch as the DNA endonuclease DNase I.

The term “sample,” as used herein, is used in its broadest sense. Asample suspected of containing a nucleic acid can comprise a cell,chromosomes isolated from a cell (e.g., a spread of metaphasechromosomes), genomic DNA, RNA, cDNA and the like.

The term “detection,” as used herein, refers to quantitatively orqualitatively identifying a nucleotide or nucleic acid within a sample.

The term “depolymerization,” as used herein, refers to the removal of anucleotide from the 3′ end of a nucleic acid.

The term “allele,” as used herein, refers to an alternative form of agene and the term “locus,” as used herein, refers to a particular placeon a nucleic acid molecule.

DETAILED DESCRIPTION OF THE INVENTION

A method of this invention is used to determine the presence or absenceof at least one predetermined (known) nucleic acid target sequence in anucleic acid sample. A nucleic acid target is “predetermined” in thatits sequence must be known to design a probe that hybridizes with thattarget. However, it should be noted that a nucleic acid target sequence,as used with respect to a process of this invention, may merely act as areporter to signal the presence of a different nucleic acid whosepresence is desired to be determined. That other nucleic acid ofinterest does not have to have a predetermined sequence. Furthermore, aprocess of the invention is useful in determining the identity of basewithin a target where only enough of the sequence is known to design aprobe that hybridizes to that target with partial complementarity at the3′-terminal region of the probe.

Such a method utilizes an enzyme that can depolymerize the 3′-terminusof an oligonucleotide probe hybridized to the nucleic acid targetsequence to release one or more identifier nucleotides whose presence orabsence can then be determined as an analytical output that indicatesthe presence or absence of the target sequence.

A nucleic acid target sequence is predetermined (or known) in that anucleic acid probe is provided to be partially or totally complementaryto that nucleic acid target sequence. A nucleic acid target sequence isa portion of nucleic acid sample with which the probe hybridizes if thattarget sequence is present in the sample.

A first step of the method is admixing a sample to be assayed with oneor more nucleic acid probes. The admixing of the first step is typicallycarried out under low stringency hybridizing conditions to form ahybridization composition. In such a hybridization composition, the3′-terminal region of the nucleic acid probe(s) (i) hybridizes withpartial or total complementarity to a nucleic acid target sequence thatmay be present in the sample; and (ii) includes an identifier nucleotidein the 3′-terminal region.

Preferably, the nucleic acid probe is designed to not hybridize withitself to form a hairpin structure in such a way as to interfere withhybridization of the 3′-terminal region of the probe to the targetnucleic acid. Parameters guiding probe design are well known in the art.

The hybridization composition is maintained under hybridizing conditionsfor a time period sufficient to form a treated sample that may containat least one predetermined nucleic acid target sequence hybridized witha nucleic acid probe.

In the event that the sample to be assayed does not contain a targetsequence to which the probe hybridizes, no hybridization takes place.When a method of the present invention is used to determine whether aparticular target sequence is present or absent in a sample to beassayed, the resulting treated sample may not contain a substrate forthe enzymes of the present invention. As a result, a 3′ terminal regionidentifier nucleotide is not released and the analytical output is at ornear background levels.

The treated sample is admixed with a depolymerizing amount of an enzymewhose activity is to release one or more identifier nucleotides from the3′-terminal region of the probe that is hybridized to the nucleic acidtarget to form a depolymerization reaction mixture. The choice of enzymeused in the process determines if a match or mismatch at the 3′-terminalnucleotide results in release of that 3′-terminal nucleotide. Furtherinformation regarding specific enzyme reaction conditions is discussedin detail hereinafter.

The depolymerization reaction mixture is maintained under depolymerizingconditions for a time period sufficient to permit the enzyme todepolymerize hybridized nucleic acid and release identifier nucleotidestherefrom to form a treated reaction mixture.

The presence or absence of released identifier nucleotides is thendetermined to obtain an analytical output. The analytical outputindicates the presence or absence of at least the one nucleic acidtarget sequence.

Processes of the invention can also be concerned with the degree ofhybridization of the target to the 3′-terminal region of the probe.Examples hereinafter show that the distinction between a matched andmismatched base becomes less notable as a single mismatch is at aposition further upstream from the 3′-terminal region position. There isvery little discrimination between a match and mismatch when a singlemismatch is ten to twelve residues from the 3′-terminal nucleotideposition, whereas great discrimination is observed when a singlemismatch is at the 3′-terminus. Therefore, when the degree ofcomplementarity (partial or total complementarity) of a nucleic acidprobe hybridized to a target nucleic acid sequence is referred to hereinin regard to an identifier nucleotide, this is to be understood to bereferring to within the 3′-terminal region, up to about ten residues ofthe 3′-terminal position.

In particular embodiments of the invention, it is desirable to include adestabilizing mismatch in or near the 3′-terminal region of the probe.In an example of such an embodiment, the goal is to determine whether anucleotide at an interrogation position is a match or a mismatch withthe target. Better discrimination between match and mismatch at theinterrogation position is observed when an intentional mismatch isintroduced about 2 to about 10 nucleotides from the interrogationposition or preferably about 2 to about 6 nucleotides from theinterrogation position.

The distinction of the analytical output between matched and mismatchednucleotides when there is more than a single base that is mismatchedwithin the 3′-terminal region can be evident even if mismatches arebeyond position 10 from the terminus, for example at position 11 and 12upstream of the 3′-terminal nucleotide. Thus, the phrases “about 10” and“3′-terminal region” are used above. The 3′-terminal region thereforecomprises the approximately 10 residues from the 3′-terminal nucleotide(or 3′ terminus) position of a nucleic acid.

The sufficiency of the time period for hybridization can be empiricallyascertained for a control sample for various hybridizing conditions andnucleic acid probe/target combinations. Exemplary maintenance times andconditions are provided in the specific examples hereinafter andtypically reflect low stringency hybridization conditions. In practice,once a suitable set of hybridization conditions and maintenance timeperiods are known for a given set of probes, an assay using thoseconditions provides the correct result if the nucleic acid targetsequence is present. Typical maintenance times are about 5 to about 60minutes.

The conditions and considerations with respect to hybridization of PCRprimers to template nucleic acid in PCR are applicable to thehybridization of nucleic acid probes to target sequences in a process ofthe invention. Such hybridization conditions are well known in the art,and are a matter of routine experimentation depending on factorsincluding the sequence of the nucleic acid probe and the target nucleicacid [sequence identity (homology), length and G+C content] molaramounts of nucleic acid present, buffer, salt content and duplex T_(m)among other variables.

Processes of the invention are sensitive and hybridization conditions oflow stringency (e.g. temperature of 0-4° C.) are sufficient, butmoderate stringency conditions (i.e. temperatures of 40-60° C.) alsopermit hybridization and provide acceptable results. This is true forall processes of the invention.

In one contemplated embodiment of the invention, the enzyme whoseactivity is to depolymerize hybridized nucleic acid to releasenucleotides from the probe 3′-terminal end is a template-dependentpolymerase. In such an embodiment, the reverse of a polymerase reactionis used to depolymerize a nucleic acid probe, and the identifiernucleotide is released when the 3′-terminal nucleotide of the nucleicacid probe hybridizes with total complementarity to its nucleic acidtarget sequence. A signal confirms the presence of a nucleic acid targetsequence that has the sequence sufficiently complementary to the nucleicacid probe to be detected by the process of the invention.

In an embodiment that uses a 3′→5′ exonuclease activity of a polymerase,such as Klenow or T4 DNA polymerase (but not limited to those twoenzymes), to depolymerize a nucleic acid probe, an identifier nucleotideis released when the 3′-terminal residue of the nucleic acid probe ismismatched and therefore there is only partial complementarity of the3′-terminus of the nucleic acid probe to its nucleic acid targetsequence. In this embodiment, to minimize background, the hybrid istypically purified from the un-annealed nucleic acid prior to the enzymereaction, which releases identifier nucleotides. A signal confirms thepresence of a nucleic acid target sequence that is not totallycomplementary to the nucleic acid probe.

In an embodiment that uses a 3′→5′ exonuclease activity of ExonucleaseIII to depolymerize a nucleic acid probe, an identifier nucleotide isreleased when the 3′-terminal residue of the nucleic acid probe ismatched to the target nucleic acid. A signal confirms the presence of anucleic acid target that is complementary at the released identifiernucleotide.

It is thus seen that hybridization and depolymerization can lead to therelease of an indicator nucleotide or to little or no release of such anucleotide, depending upon whether the probe:target hybrid is matched ormismatched at the 3′-terminal region. This is also dependent on the typeof enzyme used and the type of end, matched or mismatched, that theenzyme requires for depolymerization activity.

The magnitude of a contemplated analytical output under definedconditions is dependent upon the amount of released nucleotides. Wherean identifier nucleotide is released, an analytical output can beprovided that has a value greater than background. Where an identifiernucleotide is not released either because the target sequence was notpresent in the original sample or because the probe and depolymerizingenzyme chosen do not provide release of a 3′-terminal nucleotide whenthe target is present, or if the match/mismatch state of the 3′-terminalnucleotide did not match that required for the enzyme used to release a3′-terminal nucleotide, the analytical output is substantially at abackground level. Further discussion of computation of background levelscan be found in the Examples, e.g. Example 1.

Depolymerization reactions and enzymes useful in such reactions arediscussed below.

Depolymerization

Nucleic acid polymerases generally catalyze the elongation of nucleicacid chains. The reaction is driven by the cleavage of a pyrophosphatereleased as each nucleotide is added. Each nucleoside-5′-triphosphatehas three phosphate groups linked to carbon five of the ribose ordeoxyribose sugar. The addition of a nucleotide to a growing nucleicacid results in formation of an internucleoside phosphodiester bond.This bond is characterized in having a 3′ linkage to carbon 3 of riboseor deoxyribose and a 5′ linkage to carbon 5 of ribose or deoxyribose.Each nucleotide is added through formation of a new 3′→5′ linkage, sothe nucleic acid strand grows in a 5′ to 3′ direction.

Depolymerization in its strictest sense means the reverse ofpolymerization so that in the present context, an internucleotidephosphodiester bond is broken between the two 3′-terminal bases in thepresence of pyrophosphate and a polymerase enzyme to form a nucleic acidthat is one nucleotide shorter and a nucleoside triphosphate. A somewhatmore encompassing definition is contemplated here. In accordance withthat definition, the 3′-terminal nucleotide is removed from a nucleicacid in a reaction catalyzed by an enzyme, but the nucleotide formed canbe a monophosphate and pyrophosphate is not always required.

The former reactions are referred to herein as pyrophosphorolysisreactions whereas the latter reactions are referred to as exonucleasereactions. These two types of depolymerization are discussed below.

It is to be understood that the depolymerization reaction of interest inthe invention is that depolymerization occurring in the 3′-terminalregion of the nucleic acid probe. This depolymerization reactionreleases identifier nucleotide, as discussed herein.

A. Pyrophosphorolysis

In some embodiments of the present invention, a method comprisesdepolymerizing the nucleic acid (NA) at a 3′-terminal nucleotide byenzymatically cleaving the terminal internucleoside phosphodiester bondin the presence of pyrophosphate, or an analogue thereof, to form an XTPas illustrated by the following reaction on double-stranded DNA having a5′ overhang: 5′ . . . TpApCpGpGpCpT-3′OH 3′ . . . ApTpGpCpCpGpApCpTp-5′              ↓ enzyme + PPi 5′ . . . TpApCpGp-3′OH 3′ . . .ApTpGpCpCpGpApCpTp-5′ + dGTP + dCTP + dTTP

Several polymerases are known to catalyze the reverse of thepolymerization process. This reverse reaction is called“pyrophosphorolysis.” The pyrophosphorolysis activity of DNA polymerasewas demonstrated by Deutscher and Kornberg, J. Biol. Chem., 244:3019-28(1969). Other template-dependent nucleic acid polymerases capable ofpyrophosphorolysis include, but are not limited to, DNA polymerase α,DNA polymerase β, T4 DNA polymerase, Taq polymerase, Tne polymerase, Tnetriple mutant polymerase, Tth polymerase, Tvu polymerase, Athpolymerase, Bst polymerase, E. coli DNA polymerase I, Klenow fragment,Klenow exo minus (exo−), AMV reverse transcriptase, RNA polymerase andMMLV reverse transcriptase. However, not all polymerases are known topossess pyrophosphorolysis activity. For example, poly(A) polymerase hasbeen reported to not catalyze pyrophosphorylation. (See Sippel, Eur. J.Biochem. 37:31-40 (1973)).

A mechanism of pyrophosphorolysis has been suggested for RNA polymerase.Although understanding of the mechanism is not necessary to use thepresent invention, it is believed that the partial transfer of a Mg²⁺ion from the attacking pyrophosphate to the phosphate of theinternucleoside phosphodiester bond of the RNA can increase thenucleophilic reactivity of the pyrophosphate and the electrophilicity ofthe diester as described in Rozovskaya et al., Biochem. J., 224:645-50(1984). The internucleoside phosphodiester bond is enzymatically cleavedby the addition of pyrophosphate to the nucleoside 5′ phosphate and anew phosphodiester bond is formed between the pyrophosphate and thenucleoside monophosphate.

The pyrophosphorolysis reaction can be summarized as follows:NA_(n)+PP_(i)→NA_(n-1)+XTP  Reaction 1wherein NA is a nucleic acid, n is the number of nucleotide bases,PP_(i) is pyrophosphate and XTP is either a DNTP molecule or NTPmolecule. The reaction can then be repeated so as to produce at leasttwo XTP molecules. It should be noted that the reaction can be repeatedon the same nucleic acid molecule or on a plurality of different nucleicacid molecules.

In a preferred embodiment in the case of the reverse of polymeraseactivity (pyrophosphorolysis), a preferred substrate is a DNA probehybridized to a nucleic acid target sequence with total complementarityat its 3′-terminus, including an identifier residue at the 3′-terminalregion. In an example of this preferred embodiment, when the nucleicacid probe is hybridized to a nucleic acid target sequence such thatthere is one base mismatch at the 3′-terminal nucleotide of the nucleicacid probe, the nucleic acid probe is inefficiently depolymerizedthrough the reverse polymerization reaction. Thus, such a substrate isnot an ideal substrate for depolymerization.

The non-ideality of the substrate for depolymerization via a reverse ofthe polymerization reaction is recognized with a single base mismatch asfar in as about 10 residues from the 3′-terminus of the nucleic acidprobe. With a single base mismatch 12 residues from the 3′-terminus ofthe probe, the depolymerization reaction can occur to approximately thesame extent as when there is no mismatch and the nucleic acid probe istotally complementary to the nucleic acid target sequence.

It is thus contemplated that the reactivity of the depolymerizationreaction is a continuum that is related to the efficiency of thesubstrate. A partially complementary hybrid is a less efficientdepolymerization substrate than a totally complementary hybrid for thereverse of a polymerization reaction. It is contemplated that thisdifferential reactivity be used to enhance the discrimination betweenmatches and mismatches at certain positions (e.g. an interrogationposition). When a substrate hybrid is totally complementary, it willgive a fairly high analytical output. A mismatch can be intentionallyintroduced to destabilize the substrate hybrid. Such a destabilizationcan increase the difference in analytical output between basessubstituted at an interrogation position that is different from thedestabilizing base position.

Several chemical compounds are known in the art to be substitutable forpyrophosphate in pyrophosphorolysis reactions. Rozovskaya, et al.,Biochem. J., 224:645-650 (1984). Exemplary compounds and their releasednucleotide product are shown in the table below, along with thenucleotide product (where the ribonucleoside or deoxyribonucleoside isdenoted “Nuc”) of pyrophosphorolysis. PPi Analog PyrophosphorylationProduct

Preferred reaction mixtures for depolymerization by pyrophosphorolysis,including suitable buffers for each nucleic acid polymerase analyzed,are described in greater detail in the Examples. Typically, under theseconditions, sufficient NTP or dNTP is released to accurately detect orassay extremely low amounts of nucleic acids (e.g., about 5-1000picograms). ATP can be produced by conversion from XTP by an enzyme suchas NDPK (in the presence of ADP) prior to analysis or the ATP can befurther amplified prior to analysis.

Even though the preferred reaction conditions for polymerization anddepolymerization by pyrophosphorolysis are similar, the rates of thesereactions can vary greatly. For example, AMV and RLV reversetranscriptases catalyze pyrophosphorolysis under optimal conditions at arate of about fifty- to one hundred-fold less than polymerization asdemonstrated in Srivastavan and Modak, J. Biol. Chem., 255(5) :2000-04(1980). Thus, the high efficiency of the pyrophosphorolysis reaction wasunexpected, and appears to be associated with extremely low levels ofDNA substrate, in contrast to previous DNA pyrophosphorolysis studiesconducted using much greater amounts of DNA.

Although not wishing to be bound by theory, a possible explanation forthis effect might also be that the molar concentrations of freedeoxyribonucleoside triphosphates produced at very low DNA levels wouldbe predicted to be very low. Indeed, these levels are expected to be farbelow the Michaelis constant (K_(m)) of the enzyme. Thus,reincorporation of released dNTPs would be expected to be vanishinglysmall.

The pyrophosphorolysis activity of different nucleic acid polymerasesalso varies. For example, T4 polymerase and Tne DNA polymeraze possessvery high pyrophosphorolysis activity as measured by a luciferase assayfor ATP produced by pyrophosphorolysis. Pyrophosphorolysis using T4polymerase resulted in about a 10 fold increase in light production ascompared to MMLV-RT and a 4 fold increase in light production ascompared to Taq polymerase.

During the development of the invention disclosed in the parentapplication, it was discovered that the detection of some types ofnucleic acids at low picogram levels is generally enhanced byfragmenting or partially digesting the nucleic acid. Preferably,fragmentation is accomplished by sonication or restriction enzymedigestion of the nucleic acid in order to provide a plurality of smallernucleic acid fragments. Although an understanding of the mechanism isnot necessary in order to practice the present invention, this stepprobably enhances detection because the pyrophosphorolysis reaction onlyproceeds from the nucleic acid ends. By providing a greater number ofnucleic acid ends, more reactions are allowed to occur at any one time.

It should be noted that DNA ends can be present within a molecule aswell as at the end of a linear DNA fragment. For example, polymerasescan catalyze pyrophosphorolysis from a gap or a nick in a DNA segment.The type of enzyme and substrate used for pyrophosphorolysis reactionsdetermine whether fragmentation is necessary.

The type of DNA end resulting from restriction enzyme digestion alsoaffects the pyrophosphorolysis activity of different nucleic acidpolymerases. For example, Klenow exo−, MMLV-RT and Taq polymerasecatalyze pyrophosphorolysis of DNA fragments with 5′-overhangs and withblunt-ends, but have little or no pyrophosphorolysis activity with3′-overhangs. In contrast, T4 DNA polymerase catalyzes both 3′- and5′-end overhang and blunt-end mediated pyrophosphorolysis. Thus, T4 DNApolymerase is a preferred enzyme for pyrophosphorolysis of a hybrid witha 3′-overhang. When other nucleic acid polymerases are utilized forpyrophosphorolysis of restriction enzyme treated DNA, it is contemplatedthat care is taken to match the end specificity of the polymerase withthe type of end created by the restriction endonuclease. Such care iswell within the skill of those in the art.

Tabor and Richardson, J. Biol. Chem. 265 (14):8322-28 (1990) reportedunwanted pyrophosphorolysis mediated by T7 DNA polymerase-catalyzed DNAsequencing by the chain termination method. Those authors note that,even at the most sensitive sites, the rate of unwantedpyrophosphorolysis is at least 100,000 times slower than the rate ofpolymerization.

By definition, DNA sequencing is directed to ascertaining an unknown DNAsequence, rather than the detection of a known DNA sequence. In DNAsequencing by the chain termination method, oligonucleotide primers areextended by T7 DNA polymerase supplied with exogenous dNTPs and dideoxyNTPs. When a dideoxy NTP is incorporated into an elongating primer, nofurther polymerization can take place. These dideoxy-terminatedfragments are then resolved on a DNA sequencing gel. However, in certaininstances unwanted pyrophosphorolysis removes a 3′-terminaldideoxynucleotide from the elongated primer, which allows T7 DNApolymerase to catalyze additional polymerization. This additionalpolymerization leads to the degradation (loss) of specificdideoxynucleotide-terminated fragments on DNA sequencing gels. In otherwords, the resulting DNA sequencing gel will exhibit “holes” or gapswhere the DNA sequence cannot be determined.

Tabor and Richardson, above, noted that when dNTPs are present in highconcentrations, these pyrophosphorolysis sites occur once in severalthousand nucleotides. Those authors have identified a canonicalsequence, 5′ dIdAdN₁ddN₂ 3′, which is especially sensitive topyrophosphorolysis when dITP is substituted for dGTP. This unwanted T7DNA polymerase-mediated pyrophosphorolysis reaction can be avoided bythe addition of pyrophosphatase, which eliminates PP_(i) from the DNAsequencing reaction mixture. Pyrophosphatase, it is reported, eliminatesthe gaps in a DNA sequencing gel, permitting the accurate determinationof a DNA sequence using T7 DNA polymerase-mediated dideoxy sequencing.

The present invention, in contrast, seeks to exploit DNApolymerase-mediated pyrophosphorolysis, by optimizing conditions forthis reverse reaction to take place. The present invention is directedto the detection of a known sequence in a target nucleic acid, ratherthan ascertaining an unknown nucleic acid sequence using thepolymerization activity of T7 DNA polymerase.

The pyrophosphorolysis reported by Tabor and Richardson cannot detectthe presence of a specific nucleic acid sequence. In fact, DNAsequencing by the dideoxy method relies upon the incorporation ofdideoxy nucleotides into an elongating primer. The T7 DNApolymerase-mediated pyrophosphorolysis reported by those authors isequally random, although there is reported a preference for theabove-mentioned canonical sequence. According to Tabor and Richardson,in the absence of pyrophosphatase, one would only note gaps in a DNAsequencing gel, and those gaps would not provide any information as tothe DNA sequence at those gaps. There is accordingly no method disclosedfor identifying the release of 3′dideoxy nucleotides by the reported T7DNA polymerase-mediated pyrophosphorolysis.

Further, it is contemplated that the type of polymerase used in thepyrophosphorolysis reaction is matched to the correct nucleic acidsubstrate in order to produce the best results. In general, DNApolymerases and reverse transcriptases are preferred for depolymerizingDNA, whereas RNA polymerases are preferred for depolymerizing RNA.Reverse transcriptases or DNA polymerases with reverse transcriptaseactivity are preferred for depolymerizing RNA-DNA hybrids.

In the parent application, it was surprisingly determined that poly(A)polymerase can catalyze pyrophosphorolysis, even though no such reactionhad been previously reported. Indeed, poly(A) polymerase has been widelyreported to not catalyze pyrophosphorolysis. (See e.g., Sippel, Eur. J.Biochem., 37:31-40 (1973) and Sano and Feix, Eur. J. Biochem., 71:577-83(1976)). In these preferred embodiments of the invention disclosed inthe parent application, the manganese chloride present in the previouslyreported buffers is omitted, the concentration of sodium chloride isdecreased, and the pH value is lowered from about 8.0 to about 7.5.Furthermore, the poly(A) polymerase pyrophosphorolysis reaction buffercontains about 50 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 50 mM NaCl, and 2 mMNaPP_(i) (sodium pyrophosphate).

It is important to note that the depolymerization reaction is thereverse of the polymerization reaction. Therefore, as increasing amountsof free nucleoside triphosphates are produced by depolymerization, astate of equilibrium can theoretically be attained in whichpolymerization and depolymerization reactions are balanced.Alternatively, where small amounts of nucleic acid are detected, thereaction can go essentially to completion without reaching equilibrium,(i.e., the nucleic acid target is depolymerized into its constituentsubunit nucleotides by greater than 50%). This factor is important inquantitative assays because the total amount of nucleotides released isproportional to the amount of signal generated in the detection assay.

When used for qualitative detection of nucleic acid, as long as athreshold level of nucleotides is produced, it is not necessary that thereaction reach equilibrium or go essentially to completion. In preferredembodiments, the mixture of nucleoside triphosphate molecules producedby depolymerization is preferably converted to ATP as described below.For either quantitative or qualitative detection, a detectable thresholdATP concentration of approximately 1×10⁻¹² molar in 100 μl of sample ispreferably provided for detection of light in a typical luciferaseassay.

In some preferred embodiments, oligonucleotide probes are typicallyutilized at about 100 ng to about 1 μg per 20 μl depolymerizationreaction. That amount provides a probe to target weight ratio of about200:1 to about 1,000:1.

In a preferred embodiment of the present invention, nucleic acidpolymerase and pyrophosphate (PP_(i)) or an analogue thereof, are addedto a hybridized sample containing from less than about 100 μg of targetnucleic acid, to less than about 10 pg of nucleic acid. Typical targetnucleic acids are present at about 1 to about 5 ng in the sample to beassayed, with a target nucleic acid length of about 30 to about 1000 bpbeing preferred.

Next, the hybridized nucleic acid is degraded (depolymerized) bypyrophosphorolysis, releasing free NTPs or dNTPs. Enzymes useful in thepyrophosphorolysis reaction include, but are not limited to, those notedpreviously such as the following polymerases: AMV reverse transcriptase,MMLV reverse transcriptase, DNA polymerase alpha and beta, Taqpolymerase, Tne polymerase, Ath polymerase, Tvu polymerase, Tne triplemutant polymerase, T4 DNA polymerase, E. coli DNA polymerase I, Klenowfragment, Klenow exo minus, Tth polymerase, and poly(A) polymerase.

Most preferably, Klenow exo minus (Klenow exo−) or Tne triple mutantpolymerase is utilized for DNA pyrophosphorolysis reactions because oftheir efficient utilization of 5′ overhanging DNA ends.

When using enzymes that utilize 5′ overhang substrates, it is preferredthat the 3′ end of the target nucleic acid extends beyond the 5′ end ofthe nucleic acid probe. In this way, the only 5′ overhang substrate isthat where the 5′ end of the target nucleic acid overhangs the 3′terminal region of the nucleic acid probe. An alternative method oflimiting depolymerization to the nucleic acid probe is chemicalmodification of the ends of other nucleic acids in the sample, such as,for example, making a phosphorothioate linkage at the 3′-terminus of thetarget nucleic acid.

A depolymerizing enzyme is preferably present in an amount sufficient todepolymerize a hybridized target:probe. That amount can vary with theenzyme used, the depolymerization temperature, the buffer, and the like,as are well-known in the art. For a typical reaction carried out in a 20μL volume, about 0.25 to about 1 unit (U) of an enzyme such as Klenowexo− is used. About 1 to about 5 U of the thermostable enzymes are usedfor depolymerization at elevated temperatures.

Luciferase, which is part of the preferred ATP detection system, isinhibited by PP_(i). In preferred embodiments, care is taken to avoidtransferring a highly inhibiting amount of PP_(i) to the ATP detectionreaction. Preferably, the amount of PP_(i) carried over to the ATPdetection reaction results in a concentration of PP_(i) in theluciferase detection reaction of less than about 100 μM, although lessthan about 10 μM is desirable. Therefore, the amount of PP_(i) utilizedin the pyrophosphorolysis reaction is determined by the size of thealiquot that is taken for use in the luciferase detection system. It iscontemplated that the aliquot size can vary depending upon the testsystem used, but the amount of PP_(i) transferred or carried over to theluciferase detection reaction corresponds to the PP_(i) concentrationparameters described above, so that the concentration of PP_(i) is atleast below about 100 μM, and preferably below about 10 μM.

In one preferred embodiment of the invention, the enzyme whose activityis to depolymerize is a template-dependent polymerase. Thedepolymerization reaction is a reverse of the polymerization reaction.In a contemplated embodiment, the polymerization reaction is reversed inthe presence of pyrophosphate in a reaction referred to aspyrophosphorolysis.

In some preferred embodiments, the reaction conditions are preferablyadjusted to further favor depolymerization of a nucleic acid probe thatis hybridized with its target nucleic acid sequence by providing ahigher concentration of nucleic acid probe than its target nucleic acidsequence.

One strategy to favor the depolymerization of a probe:target hybrid isthat the probe be in excess over the nucleic acid target in thehybridization step after denaturing of duplex target nucleic acid.

Another strategy to favor the depolymerization of a probe:target hybridis to isolate only the strand of nucleic acid target to which the probeis complementary. There are several techniques that can be used toachieve this end.

In one technique, phosphorothioate linkages are utilized at the5′-terminus of a target nucleic acid amplifying primer sequence, e.g.,at the 1 to about 10 5′-most residues. Upon PCR amplification of thetarget, the phosphorothioate linkages of the primer become incorporatedinto the amplified target nucleic acid as part of one of a pair ofcomplementary strands. Treatment of the double-stranded resultingmolecule with T7 polymerase exonuclease 6 removes thenon-phosphorothioate-containing strand. This technique is illustrated indetail in the Examples hereinafter.

In another technique, strand isolation can be accomplished by amplifyingthe target nucleic acid using PCR primers incorporated into the extendednucleic acid strand (with which a nucleic acid probe useful herein isdesigned to hybridize) that are not labeled, whereas primers for thecomplementary strand are labeled, such as with biotin. Then, theamplified nucleic acid is denatured and added to streptavidin linked toa solid support. A useful material is Streptavidin MagneSphere®paramagnetic particles (Promega, Z548A), where a magnet can be used toseparate the desired target nucleic acid strand from its biotinylatedcomplementary strand.

B. Exonuclease Digestion

In other embodiments of the present invention, a method comprisesdepolymerizing the nucleic acid at a 3′-terminal nucleotide byenzymatically cleaving the terminal internucleoside phosphodiester bondto form an XMP as illustrated by the following reaction ondouble-stranded DNA having a 5′-overhang: 5′ . . . GpCpTpApApGpT-3′OH 3′. . . CpGpApTpTpCpApCpTp-5′               ↓ enzyme 5′ . . . GpCpTpA-3′OH3′ . . . CpGpApTpTpCpApCpTp-5′ + dAMP + dGMP + dTMP

For example, such a hydrolysis reaction can be catalyzed by Klenow orExonuclease III in the presence or absence of NTPs.

In some embodiments (e.g., quantitative assays for nucleic acids), thedepolymerizing step is repeated essentially to completion or equilibriumto obtain at least two nucleotide molecules from a strand of minimallythree nucleotides in order to increase detection sensitivity. Inalternative embodiments, (e.g., qualitative detection of DNA), thedepolymerizing step need not be repeated if there are sufficient nucleicacid molecules present to generate a signal.

In another embodiment of the present invention, terminally mismatchedhybridized nucleic acid probes are first depolymerized into NMP or dNMPby exonuclease digestion according to the following reaction:NA_(n)+H₂O→NA_(n-1)+XMP  Reaction 2

wherein NA_(n) is a nucleic acid, XMP is either a dNMP or NMP, and n isthe number of nucleotides in the nucleic acid.

This depolymerization reaction is shown more specifically below in thefollowing reaction on double-stranded DNA having a 5′-overhang andmismatched bases at the 3′-terminus: 5′ . . . CpTpApApGpC-3′OH 3′ . . .GpApTpTpCpApCpTp-5′               ↓ enzyme 5′ . . . CpTpApApG-3′OH 3′ .. . GpApTpTpCpApCpTp-5′ + dCMP

For example, such a depolymerization reaction can be catalyzed bybacteriophage T4 polymerase in the absence of NTPs. In preferredembodiments, the released nucleotides, XMPs, are produced by nucleasedigestion.

Nuclease digestion can be accomplished by a variety of nucleases thatrelease a nucleotide with a 5′ phosphate, including Si nuclease,nuclease BAL 31, mung bean nuclease, exonuclease III and ribonuclease H.Nuclease digestion conditions and buffers are known in the art.Nucleases and buffers for their use are available from commercialsources.

In the biosynthesis of purine and pyrimidine mononucleotides,phosphoribosyl-1-pyrophosphate (PRPP) is the obligatoryribose-5′-phosphate donor. PRPP itself is formed in a reaction catalyzedby PRPP synthetase through the transfer of pyrophosphate from ATP toribose-5′-phosphate. This reaction is known to be reversible asdescribed in Sabina et al., Science, 223:1193-95 (1984).

In some embodiments of the present invention, the NMP or dNMP producedby nuclease digestion is preferably converted directly to NTP or dNTP bythe enzyme PRPP synthetase in the following reaction:XMP+PRPP→XTP+ribose-5′-PO₄  Reaction 3

wherein XMP is either AMP or dAMP, and XTP is either ATP or dATP.Preferably, this reaction produces a threshold ATP concentration ofapproximately 1×10⁻¹² M in 100 μl of sample.

In this reaction, the pyrophosphate group of PRPP is enzymaticallytransferred to XMP molecules, forming XTP molecules. Examples ofsuitable reaction conditions and buffers are set forth elsewhere herein.

Utilization of the PRPP reaction in the nucleic acid detection system ofthe present invention has advantages over previously reported methods.For example, only one step is necessary to convert an AMP or dAMP to ATPor dATP, thereby simplifying the detection system. In addition,contamination of the detection reaction with exogenous ATP, ADP, or AMPis less likely using methods of the present invention, as compared topreviously reported methods.

In an embodiment wherein the depolymerizing enzyme exhibits 3′→5′exonuclease activity, the substrate is a double-stranded orsingle-stranded nucleic acid having a 3′-hydroxyl terminus. Enzymeshaving 3′→5′ exonuclease activity that are useful in a process of theinvention include E. coli DNA polymerase I, Klenow fragment andbacteriophage T4 DNA polymerase. E. coli DNA polymerase I holoenzyme isnot preferred in a process of the invention because it is preferable toavoid the 5′→3′ exonuclease activity that degrades probe:target hybridsregardless of the degree of hybridization at the 3′-terminus.Bacteriophage λ exonuclease has only 5′→3′ exonuclease activity, so itis not a contemplated enzyme. Similarly, Taq DNA polymerase has a verylow level of 3′→5′ exonuclease activity. Exonuclease III (Exo III) has3′ exonuclease activity on blunt-ended substrates or those having5′-overhangs or nicks with 3′-hydroxyl groups, and is thus useful in aprocess of the invention for depolymerizing hybrids with matched 3′terminal nucleotides. However, Exo III is not limited to hybrids havingonly partially complementary 3′-termini, it requires a double strandedend, i.e. a matched terminal nucleotide.

In an embodiment of the invention where the enzyme's activity is a 3′→5′exonuclease activity, the hybridized nucleic acid probe is depolymerizedfrom its 3′-terminal nucleotide. In a preferred embodiment in the caseof a 3′→5′ exonuclease activity of a polymerase, the preferred substrateis a nucleic acid probe hybridized to a nucleic acid target sequencewith partial complementarity at its 3′-terminal region, most preferablywith a mismatch at its 3′-terminal residue that is an identifiernucleotide.

A contemplated method is particularly useful in a multiplex assayenvironment in which a plurality of probes is utilized to determinewhether one or more of a plurality of predetermined nucleic acidsequences is present or absent in a sample. A particularly useful areafor such multiplex assays is in screening assays where the usualanalytical output indicates that the sought-after gene is absent.

In one illustrative embodiment, a nucleic acid sample is screened forthe presence of a plurality of predetermined mutant genes. In thisembodiment, the mutants usually are not present and the analyticaloutput is, for example, at about background levels except where amutation is present. In another embodiment, a plurality of samples isexamined for the presence or absence of microbe-specific genes. Here,again, where a population of healthy individuals, animals, or presumablysterile food is sampled, the absence of the sought-after genes providesan analytical output that is about background levels, and only in therare instance does a greater than the background output appear.

In a multiplexed embodiment of the above process, the sample is admixedwith a plurality of different nucleic acid probes, preferably afteramplification of the multiple nucleic acid targets as needed. In thisembodiment of the invention, the analytical output for a certain resultwith one of the probes is distinguishable from the analytical outputfrom the opposite result with all of the probes.

In preferred embodiments, the ATP produced via NDPK conversion ofreleased nucleotides in the presence of ADP is detected by a luciferasedetection system or an NADH detection system. In still anotherembodiment of the present invention, the pyrophosphate transferring stepand the phosphate transferring step are performed in a single potreaction. In other preferred embodiments, if increased sensitivity isrequired, the ATP molecules can be amplified.

In a contemplated multiplex embodiment, information about the presenceor absence of a plurality of nucleic acid target sequences is determinedusing a process of the invention on a single nucleic acid sample, byadmixing the sample with a plurality of nucleic acid probes for thevarious nucleic acid targets.

In a first multiplex embodiment of the invention, the analytical outputobtained when at least one of the nucleic acid probes hybridizes withpartial complementarity to its target nucleic acid sequence is greaterthan the analytical output when all of the nucleic acid probes hybridizewith total complementarity to their respective nucleic acid targetsequences. Preferably, in such an embodiment, the enzyme whose activityis to depolymerize hybridized nucleic acid to release nucleotidesexhibits a 3′→5′-exonuclease activity, depolymerizing hybridized nucleicacids having one or more mismatched bases at the 3′-terminus of thehybridized probe.

In a second multiplex embodiment of the invention, the analytical outputobtained when at least one of said nucleic acid probes hybridizes withpartial complementarity to its target nucleic acid sequence is less thanthe analytical output when all of the nucleic acid probes hybridize withtotal complementarity to their respective nucleic acid target sequences.Preferably, in such an embodiment, the enzyme whose activity is todepolymerize hybridized nucleic acid to release nucleotides is atemplate-dependent polymerase.

In a third multiplex embodiment of the invention, the analytical outputobtained when at least one of said nucleic acid probes hybridizes withtotal complementarity to its nucleic acid target sequence is greaterthan the analytical output when all of the nucleic acid probes hybridizewith partial complementarity to their respective nucleic acid targetsequences. Preferably, in such an embodiment, the enzyme whose activityis to depolymerize hybridized nucleic acid to release nucleotides is atemplate-dependent polymerase.

In a fourth multiplex embodiment of the invention, the analytical outputobtained when at least one of said nucleic acid probes hybridizes withtotal complementarity to its target nucleic acid sequence is less thanthe analytical output when all of the nucleic acid probes hybridize withpartial complementarity to their respective nucleic acid targetsequences. Preferably, in such an embodiment, the enzyme whose activityis to depolymerize hybridized nucleic acid to release nucleotidesexhibits a 3′→5′-exonuclease activity, depolymerizing hybridized nucleicacids having one or more mismatched bases at the 3′-terminus of thehybridized probe.

Analytical Output

The analytical output is obtained by detection of the releasedidentifier products, either the released nucleotides or the remainder ofthe probe. Exemplary detection systems include the light emittingluciferase detection system, the NADH light adsorption detection system(NADH detection system), fluorescence emissions and mass spectrometry.These detection systems are discussed hereinbelow.

The fact that nucleotides were released (a qualitative determination),or even the number of nucleotides released (a quantitativedetermination) can be deduced through examination of the probe afterdepolymerization. The determination of the size of an oligonucleotide iswell known in the art. For example gel separation and chromatographicseparations are well known. Gel imaging techniques that take advantageof fluorescence and absorbance spectroscopy as well as radiographicmethods. Mass spectrometry of oligonucleotides is also becoming morecommon.

A. Detection of ATP

Luciferase detection systems are particularly useful for detecting ATP.In the presence of ATP and oxygen, luciferase catalyzes the oxidation ofluciferin, producing light that can then be quantified using aluminometer. Additional products of the reaction are AMP, pyrophosphateand oxyluciferin.

In particularly preferred embodiments, ATP detection buffer referred toas L/L reagent (Promega, FF2021) is utilized. In some embodiments,Luciferase Assay Reagent (LAR) buffer (Promega, E152A) is used insteadof L/L reagent. Preferably, about 5 to 10 ng of luciferase are used inthe reaction. Although it is not intended that the present invention belimited to a specific concentration of luciferase, greater amounts ofluciferase have a tendency to increase non-specific background.

It is contemplated that in some embodiments, the dNTPs or NTPs producedby pyrophosphorolysis or nuclease digestion are converted to XTP, whichcan then be used directly as substrate for luciferase, permittingdetection of the nucleic acid. However, the preferred substrate forluciferase is ATP, as demonstrated by Moyer and Henderson, Anal.Biochem., 131:187-89 (1983). When DNA is the initial substrate, NDPK isconveniently utilized to catalyze the conversion of dNTPs to ATP by thefollowing general reaction:dNTP*+ADP→dNDP+ATP*  Reaction 4

wherein DNTP is a mixture of deoxyribonucleoside triphosphates and dNDPis the corresponding deoxyribonucleoside diphosphate. In Reaction 4, theterminal 5′-triphosphate (P*) of the dNTP is transferred to ADP to formATP.

Enzymes catalyzing this reaction are generally known as nucleosidediphosphate kinases (NDPKs). NDPKs are ubiquitous, relativelynonspecific enzymes. For a review of NDPK, see Parks and Agarwal, in TheEnzymes, Volume 8, P. Boyer Ed. (1973).

The conversion of NTPs or dNTPs to ATP by NDPK is preferablyaccomplished by adding NDPK and a molar excess of ADP over the amountsof NTPs or dNTPs expected to be produced by pyrophosphorolysis ornuclease digestion, followed by pyrophosphorylation by PRPP synthetase.The utilization of ADP requires optimization of the amount of ADP added.Too much ADP results in high background levels.

NDPK (EC 2.7.4.6) preparations from several biological sources arecommercially available from several suppliers. For example yeast NDPK isavailable from Sigma Chemical Co., St. Louis, Mo., whereas bovine NDPKis available from ICN Biochemicals, Inc., Costa Mesa, Calif. Theparticular NDPK selected for most uses described herein is typically amatter of choice.

A further embodiment of the invention, such as is used for Single TandemRepeat (STR) detection, contemplates a method for determining the numberof known repeated sequences that are present in a nucleic acid targetsequence in a nucleic acid sample. A method for determining the numberof repeated known sequences comprises the following steps. A pluralityof separately treated samples is provided. Each sample contains anucleic acid target sequence, containing a plurality of known repeatedsequences and a non-repeated region, hybridized with a nucleic acidprobe. Each nucleic acid probe contains a different number ofcomplementary known repeated sequences of alleles of the target nucleicacid, an identifier nucleotide in the 3′-terminal region and a5′-terminal locker sequence that is complementary to the non-repeatedregion of the target. A treated reaction mixture is formed by admixingeach treated sample with a depolymerizing amount of an enzyme whoseactivity is to release one or more nucleotides from the 3′-terminus of ahybridized nucleic acid probe. The treated reaction mixture ismaintained for a time period sufficient to permit the enzyme todepolymerize the hybridized nucleic acid probe and release an identifiernucleotide. The samples are analyzed for the presence or absence ofreleased identifier nucleotide to obtain an analytical output. Theanalytical output from the sample whose probe contained the same numberof sequence repeats as present in the target nucleic acid is indicativeof and determines the number of sequence repeats present in the nucleicacid target.

In one aspect of the method, the target nucleic acid is homozygous withrespect to the number of the repeated sequences at the two alleles. Inan alternative method of the invention, the target nucleic acid isheterozygous for the repeated sequences. In another method of theinvention, an identifier nucleotide is a nucleotide that is part of theregion containing a repeated sequence. In an alternative method of theinvention, an identifier nucleotide of the probe sequence is part of theregion containing a non-repeating sequence that is complementary to thatlocated in the target nucleic acid 5′ to the repeated sequences. In thislatter aspect of the method, the identifier nucleotide is present in asequence containing 1 to about 20 nucleic acids that is complementary toa non-repeating sequence of the target nucleic acid located in the probe3′ to the repeated sequences. The repeated known sequence present in anucleic acid target sequence typically has a length of 2 to about 24bases per repeat. Di- and tri-nucleotide repeats are well known in theart.

As is illustrated in the Examples that follow, it can be beneficial tocarry out a contemplated method at elevated temperatures, e.g., about50° C. to about 90° C. An above-mentioned NDPK has a very shorthalf-life at these temperatures, e.g., less than about one minute, andit is preferred to use a thermostable NDPK for reactions at theseelevated temperatures.

A particularly preferred thermostable NDPK was obtained by cloning theappropriate DNA of the thermophilic bacteria Pyrococcus furiosis (Pfu).The NDPK obtained, denoted NDPK Pfu, retains much more activity aftermaintenance at a temperature of 70° C. for a time period of 5 minutesthan did yeast NDPK, and was found to have a half-life at a temperatureof 70° C. of about 10 minutes as compared to yeast NDPK that had ahalf-life at that temperature of about 0.6 minutes. The recombinantenzyme contains 161 amino acid residues, whose sequence is provided asPf5 in SEQ ID NO:91, with a corresponding DNA sense strand sequence asPf4 in SEQ ID NO:90.

A contemplated thermostable NDPK, such as Pfu NDPK, is advantageouslyutilized in a so-called one-step or one-pot method of this invention.Here, a treated sample that may contain the predetermined nucleic acidtarget sequence hybridized with a nucleic acid probe that includes anidentifier nucleotide in the 3′-terminal region is admixed with adepolymerizing amount of an enzyme whose activity in the presence ofpyrophosphate is to release identifier nucleotides as nucleosidetriphosphates from the hybridized nucleic acid probe, adenosine 5′diphosphate (ADP), pyrophosphate and NDPK to form a treated reactionmixture. The treated reaction mixture so formed is maintained for a timeperiod sufficient to permit the enzyme to depolymerize the probe and topermit NDPK to convert the XTP present into ATP (as shown in reaction4). The amount of ATP formed is determined by the production of ananalytical output, with that output providing the indication of thepresence or absence of the presence of the target nucleic acid sequence.

Although yeast, bovine or another NDPK can be used in these reactions,it is preferred to utilize a thermostable NDPK such as the Pfu NDPKalong with a thermostable depolymerizing enzyme such as the Tne triplemutant DNA polymerase (discussed below), Bst DNA polymerase, Ath DNApolymerase, Taq DNA polymerase and Tvu DNA polymerase along with areaction temperature of about 50° C. to about 90° C. The use of thesethermostable enzymes at an above temperature can enhance the sensitivityof the method.

The Tne triple mutant DNA polymerase is described in detail in WO96/41014, whose disclosures are incorporated by reference, and its 610residue amino acid sequence is provided as SEQ ID NO:35 of thatdocument. That enzyme is referred to in WO 96/41014 as Tne M284(D323A,D389A).

Briefly, that enzyme is a triple mutant of the polymerase encoded by thethermophilic eubacterium Thermotoga neapolitana (ATCC 49049). Theamino-terminal 283 residues of the native sequence are deleted and theaspartic acid residues at positions 323 and 389 of the native sequenceare replaced by alanine residues in this recombinant enzyme. Thisrecombinant enzyme is thus a deletion and replacement mutant of thenative enzyme.

Deletion of the amino-terminal sequence removes the 5′ exonucleaseactivity of the native enzyme, whereas replacement of the two asparticacid residues removes a magnesium binding site whose presencefacilitates exonuclease activity, and this triple mutant also exhibitedno 3′ exonuclease activity relative to the recombinant native enzyme.This triple mutant enzyme exhibited a half-life at 97.5° C. of 66minutes as compared to the full length recombinant enzyme that exhibiteda half-life of only 5 minutes at that temperature.

A reaction containing NDPK contains about 0.01 to 0.50 μM ADP,preferably about 0.05 μM ADP. Various useful buffers and other reactioncomponents are set forth elsewhere. NDPK is itself present in an amountsufficient to catalyze the desired conversion of ADP to ATP. In atypical assay starting from a 20 μL depolymerization reaction, about 0.1U of NDPK are used.

Where larger volumes of reactants are used, with the target and probeconcentrations being approximately proportionately larger, the amount ofNDPK or the other enzymes discussed herein can be used in a similarlarger proportion relative to the amount discussed for the 20 μLreaction. Indeed, a 20 μL reaction has been successfully scaled downabout two fold and scaled upwardly by a factor of about 20.

As an optional step, the NTP, dNTP, or ATP generated by thepyrophosphorolysis or nuclease digestion reactions followed byappropriate treatments can be amplified to give even greatersensitivity. For example, amplification can be required when detectionsystems other than luciferase are utilized or when increased levels ofsignal are needed for detection by a less sensitive luminometer.“Amplification of NTP” refers to a continuous reaction, wherein 1 NTPgives rise to 2 NTPs, which can be cycled to yield 4 NTPs and so on.When AMP is added to feed the amplification reaction, ATP accumulates.PCT publication WO 94/25619 and Chittock et al., Anal. Biochem.,255:120-6 (1998), incorporated herein by reference, discloseamplification systems for ATP characterized by the following coupledreactions:C1+S1+E1→2C2 and 2C2+2S2+E2→2C12C1+2S1+E1→4C2 and 4C2+4S2+E2→4C14C1+4S1+E1→8C2 and 8C2+8S2+E2→8C1  Reaction 5:wherein C1 is the target compound present in a sample to be amplified,S1 is the amplification substrate, E1 is a catalytic enzyme capable ofutilizing C1 and S1 to produce C2, S2 is a high energy phosphatedonating substrate, and E2 is a catalytic enzyme capable of utilizing C2and S2 to produce C1, which then recycles through the reaction.According to this reaction scheme, each pass through the coupledreaction doubles the amount of C1, which can be subsequently detected.Patent Application GB 2,055,200 discloses an amplification systemutilizing adenylate kinase and pyruvate kinase.

In providing a coupled ATP amplification reaction for use in nucleicacid detection, two main requirements should be considered. First, E1should not be able to utilize the high energy phosphate donor utilizedby E2. If E1 can utilize the high energy phosphate donor, the ATPamplification reaction proceeds in the absence of NTP or dNTP producedas a result of pyrophosphorolysis. This results in the undesirableoccurrence of false positive results. Second, a molar excess of theadded high energy phosphate donor is preferably provided as compared tothe amount of XTP expected in the reaction. Third, E1 should be able toutilize either the NTP, dNTP, or ATP produced in step 1 bypyrophosphorolysis or nuclease digestion of the nucleic acid.

The amplification system of some preferred embodiments of the presentinvention can be characterized, as follows:XTP+AMP+E1→XDP+ADP ADP+D-P+E2→ATP  Reaction 6wherein D-P is a high energy phosphate donor and E1 and E2 are enzymescapable of catalyzing the transfer of phosphates from an XTP to AMP andfrom the D-P to ADP, respectively. The ATP so produced can reenter thereaction (i.e., as XTP) and the reaction can be repeated until thesubstrates are exhausted or equilibrium is reached, resulting in theproduction of two ATPs for every ATP supplied to or generated by thereaction. When the target XTP is any nucleoside triphosphate other thanATP, the initial pass through the cycle yields only 1 ATP that thenreenters the cycle to produce two ATP, both of which reenter the cycleto produce 4 ATP and so on. Preferably, the amplification reactionproduces a threshold ATP concentration of approximately 1×10⁻¹² Molar in100 μL of sample.

In some preferred embodiments, the XTP in the amplification system aboveis NTP or DNTP, which can preferably be ATP provided bypyrophosphorolysis (e.g., Reaction 1) or created from XTP by NDPKconversion of ADP to ATP (e.g., Reaction 4) or provided by nucleasedigestion coupled with transformation of the XMPs to XTPs (e.g.,Reaction 3) followed by NDPK conversion to ATP (e.g., Reaction 4). Itshould be appreciated, however, that when an amplification step isutilized for a DNA substrate, the step of converting dNTP to ATP isinherent in the amplification system. Therefore, a separate convertingstep is not required for the present invention.

A nucleoside monophosphate kinase (NMPK) or adenylate kinase ispreferably utilized as enzyme 1 (E1). NMPKs occur as a family, each ofwhich is responsible for catalyzing the phosphorylation of a particularNMP. Until recently, it was generally thought that ATP and dATP werepreferred phosphate donors. However, Shimofuruya and Suzuki Biochem.Intl., 26(5):853-61 (1992) recently demonstrated that at least someNMPKs can utilize other phosphate donors such as CTP and UTP. Enzyme 2(E2) is preferably NDPK or pyruvate kinase. NDPKs generally catalyze thetransfer of the terminal 5′-triphosphate of NTPs to NDPs to form NTPsfrom the NDP. Pyruvate kinase catalyzes the transfer of phosphate fromphosphoenolpyruvate (PEP) to ADP to form ATP. These enzymatic activitiesare utilized in the amplification reaction to transfer a phosphate groupfrom a high energy phosphate donor (D-P) to either ADP or an NDP.

In particularly preferred embodiments, a high energy phosphate donor(D-P) that can be used by E2 but not by E1 is used. When E2 is NDPK,dCTP or α,β-methylene adenosine 5′-triphosphate (AMP-CPP) can beutilized as D-P. When E2 is pyruvate kinase, PEP is the preferred highenergy phosphate donor.

Prior to the invention disclosed in the parent application, the abilityof NDPK to utilize these substrates at efficiencies permittingproduction of minute quantities of ATP was not known. As the recentliterature suggests that NMPK (E1) can utilize phosphate donors otherthan ATP or dATP, it is surprising that these high energy phosphatedonors utilized with NMPK meet the requirements of the amplificationreaction. The nonspecificity of adenylate kinase is also well known, andin the examples adenylate kinase is E-1, dCTP is not used as D-P.

The high energy phosphate donor and/or AMP is preferably provided in amolar excess as compared to the amount of ATP or dNTP expected to bepresent in the sample, so that the high energy phosphate donor is notrecycled at an appreciable rate. Although it is not intended that thepresent invention be limited to any particular embodiment, variousbuffers and reaction components are provided in the Examples.

B. NADH Detection

In the NADH detection system, a combination of two enzymes,phosphoglycerate kinase and glyceraldehyde phosphate dehydrogenase, isused to catalyze the formation of NAD from NADH in the presence of ATP.Because NADH is fluorescent whereas NAD is not, ATP is measured as aloss in fluorescence intensity. Examples of NADH based ATP assays aredisclosed in U.S. Pat. Nos. 4,735,897, 4,595,655, 4,446,231 and4,743,561, and UK Patent Application GB 2,055,200, all of which areherein incorporated by reference.

C. Mass Spectrometric Analysis

In one method of the invention, the presence of released nucleotides isanalyzed via mass spectrometry. In an embodiment of a method using massspectrometry, the treated reaction mixture is ionized in a manner suchthat all components of the treated reaction mixture in the molecularweight range of the released identifier nucleotides are measured. Verysmall differences in molecular weight can be detected using massspectrographic methods (different isotopes of the same atom aredetectable), so any variation from a natural nucleic acid, including asingle atom substitution (e.g. a fluorine in place of a hydrogen atom ora replacement of a hydrogen by a deuterium atom) in the identifiernucleotide gives rise to a detectable difference. Nucleic acid analogsused in methods of the invention should not interfere with either thehybridization of the nucleic acid probe or depolymerization of thehybridized probe.

Additionally, mass spectrometry can discriminate between individualnucleotides or nucleosides. For example, if the 3′-identifier nucleotideused in the instant invention was a G nucleotide, mass spectrometry canbe used to detect the release of that G nucleotide in a method of thepresent invention. Similarly, mass spectrometry can detect the releaseof an A, T or C nucleotide, based on the differences in atomic weight ofthese compounds. Thus, in a multiplexing embodiment of the presentinvention, mass spectrometry can be used to resolve the presence of oneor more of these 3′-identifier nucleotides.

In a particularly useful aspect of this embodiment, a mass spectraltechnique referred to as DIOS (desorption/ionization on silicon) wasrecently reported by Wei et al., Nature, 399:243(1999) that canaccurately perform one or multiple assays on picogram or attagramamounts using commercially available mass spectrographs adapted with aspecialized porous silicon sample well. The older, well known, MALDImass spectrographic assay techniques can also be utilized.

In an embodiment of a multiplex method using mass spectrometry, multipledifferent identifier nucleotides can be used in the various nucleic acidprobes. Using such a technique the presence of the different identifiernucleotides is direct evidence of the presence of the nucleic acidtarget sequences.

D. Fluorescence Spectroscopic Analysis

In some contemplated embodiments, the identifier nucleotide includes afluorescent label. In one embodiment when the nucleotide is afluorescent label, the analytical output is obtained by fluorescencespectroscopy. In an alternative embodiment when the nucleotide is afluorescent label, the analytical output is obtained by massspectrometry.

In a preferred embodiment of the invention, the fluorescent label ispart of a fluorescent analog of a nucleotide. Fluorescent nucleotideanalogs are widely known and commercially available from severalsources. An exemplary source is NEN™ Life Science Products (Boston,Mass.), who offer dideoxy-, deoxy-, and ribonucleotide analogs a labeledwith fluorescein, coumarin, tetramethylrhodamine, naphthofluorescein,pyrene, Texas Red®, and Lissamine™. Other suppliers include AmershamPharmacia Biotech (Uppsala, Sweden; Piscataway, N.J.) and MBI Fermentas,Inc. (Amherst, N.Y.).

An advantage to using fluorescent labels and fluorescence spectroscopyanalysis is that there are multiple different labels. Such differentlabels would be particularly useful in a multiplex embodiment of theinvention. Different fluorescent labels would be used in differentprobes, so that the detection of a particular fluorescently-labelednucleotide analog as a released identifier nucleotide could be used todeduce which nucleic acid targets are present.

For example, fluorescein has a 488 nm excitation and 520 nm emissionwavelength, whereas rhodamine (in the form of tetramethyl rhodamine) has550 nm excitation and 575 nm emission wavelength. A fluorescencedetector provides an excitation source and an emission detector. Theemission wavelengths of 520 nm and 575 nm are easily distinguishableusing fluorescence spectroscopy.

On a per molecule basis, fluorescence spectroscopy is about 10-fold moresensitive than absorbance spectroscopy. A very wide variety offluorescence spectroscopy-based detectors are commercially available forreading fluorescence values of single tubes, flow cells and multi-wellplates, among others. For example, Labsystems Multiskan models ofmicroplate readers are widely available with a spectral range of 400 to750 nm, and filters for 340, 405, 414, 450, 492, 540, 620, and 690 nm(e.g. Fisher Scientific, Pittsburgh, Pa.).

It is contemplated that a released identifier nucleotide could belabeled before or after depolymerization using cross-linking chemistrywell known in the art with commercially available reagents. For example,fluorescein isothiocyanate and rhodamine B isothiocyanate are bothavailable from Aldrich Chemical Company (Milwaukee, Wis.). References tofluorescein isothiocyanate's use in labeling biological moleculesinclude Nature, 193:167 (1962), Methods Enzymol. 26:28 (1972), Anal.Biochem., 57:227 (1974), Proc. Natl. Acad. Sci., U.S., 72:459 (1975).

It is contemplated that for many embodiments of the invention, it isuseful to separate released fluorescent identifier nucleotides fromthose bound to an oligonucleotide, such as a probe. Thus, the separationtechniques well known in the art and discussed above are useful withsuch an embodiment, including HPLC fitted with a fluorescence detector.The enhanced sensitivity of fluorescence relative to other spectroscopictechniques can be used to increase the sensitivity of a detection orquantification process of the invention.

E. Absorbance Spectroscopic Analysis

An absorbance spectrographic analysis step is contemplated to provide ananalytical output, thereby provide for the determination of the presenceor absence released identifier nucleotide, and indicate the presence orabsence of said nucleic acid target sequence. This embodimentcontemplates the chromatographic separation of a reaction mixture thathas been treated with a depolymerizing amount of an enzyme whoseactivity is to release one or more nucleotides from the 3′-terminus of ahybridized nucleic acid.

In an illustrative embodiment, a multiplexed assay for the presence ofseveral different nucleic acid target sequences in a sample is analyzedby absorbance spectroscopy. Several labeled probes to various nucleicacid target sequences are added to a nucleic acid sample. The labels onthe probes may be various nucleotide analogs, a different one for eachprobe. A depolymerizing enzyme is added, such as Klenow exo−, releasingthe labeled nucleotides and other nucleotides from the 3′-termini ofprobes hybridized to target sequences when the 3′ terminal nucleotide ismatched.

The reaction solution is loaded onto a pre-equilibrated High PressureLiquid Chromatography (HPLC) column and eluted under conditions thatseparate the nucleotide analogs from the natural nucleotides. Usefulmedia for chromatographic separation of nucleotides, bases, andnucleosides include reverse phase media, such as a reverse phase C18column or ODS-80T_(M) or ODS-120T TSK-GEL by TosoHaas (Montgomeryville,Pa.), anion exchange media, such as DEAE-25SW or SP-25W TSK-GEL byTosoHaas (Montgomeryville, Pa.), or affinity media, such as Boronate-5PWTSK-GEL by TosoHaas (Montgomeryville, Pa.). Example 65 illustrates anembodiment of the present invention using HPLC.

The HPLC column is fitted with an absorbance detector to monitor thecolumn effluent. Hence, “absorbance spectroscopy” for this type ofanalysis. Typical wavelengths for monitoring HPLC detection ofnucleotides are 250 nm, 260 nm and 280 nm. Such separations ofnucleotides and nucleotide analogs are well known in the art. Revich etal., J. Chromatography, 317:283-300 (1984), and Perrone & Brown, J.Chromatography, 317:301-310 (1984) provide examples of the HPLCseparation of dNTPs.

Identification of the separated nucleotide analogs can be accomplishedby comparison of the retention times (as monitored by absorbance ofeffluent at various times) of standards of the nucleotide analogsseparated on the same HPLC column under the same conditions.Alternatively, the identity of the nucleotide analogs collected inseparate fractions (as determined by continually monitoring theabsorbance of the column effluent) can be determined by other standardanalytical methods, such as nuclear magnetic resonance or atomicanalysis (H,C,N).

In this illustrative example using depolymerization with Klenow exo−,the presence of a released identifier nucleotide from a particular probeindicates the presence of the target sequence that hybridize with thatprobe.

In an alternative embodiment, the released nucleotides from adepolymerization reaction mixture are separated on a gas chromatographfitted with an absorbance detector to monitor column effluent.

Coupled Reactions

In some embodiments, certain of the above reactions can be performed assingle pot reactions. A “single pot reaction” is a reaction wherein atleast two enzymes (i.e., E1 and E2) with catalytic activity are presentin the same reaction mix and act on one or more substrate(s) (i.e., S1and S2). In some embodiments, the reactions catalyzed by the enzymesoccur simultaneously where E1 acts on S1 and E2 acts on S2.Alternatively, the reactions catalyzed by E1 and E2 can occur in astep-wise or coupled manner (e.g., where E1 acts on Si to produce anintermediate S2_(i) and E2 then acts on S2_(i)). Of course, in yet otherembodiments, such a coupled reaction can also be essentiallysimultaneous.

The ability to utilize combinations or mixtures of the enzymes of thepresent invention in single pot reactions is surprising, in light of theextremely low levels of nucleic acid detection that are achieved usingthe present invention. This low level detection is possible even thoughsome enzymes are used under suboptimal conditions. As previouslydescribed, it was found to be necessary to optimize the concentration ofPP_(i) utilized in the pyrophosphorolysis reactions to minimizeinhibition of luciferase. Therefore, aliquots from the NMP-, dNMP-,NTP-, dNTP- and ATP-producing reactions can be directly added to L/LReagent for luciferase detection without any purification of thereaction products. The luciferase reaction is not poisoned or otherwisequenched by the components of the reactions. This desirable featurepermits automation and high throughput analysis with a minimal amount oftime and effort, and it also permits great flexibility in the design ofthe overall detection schemes. However, it is not intended that thepresent invention be limited to any particular reaction condition,reagents, or embodiments.

In some preferred embodiments, the pyrophosphorolysis reaction producingDNTP and the NDPK catalyzed reaction in which the NTPs or dNTPs areconverted to ATP are performed in a single pot reaction in the nucleicacid polymerase buffer in these embodiments. NDPK activity is sufficientto convert dNTP to ATP, even though the polymerase buffer conditions aresuboptimal for NDPK activity.

The polymerase enzyme and NDPK can both be present initially in thereaction, or the NDPK can be added directly to the reaction after anincubation period sufficient for the production of NTP or dNTP.Alternatively, a nucleic acid polymerase and NDPK can be provided in thesame vessel or mixture for use in the reactions described above. Themixture preferably contains the nucleic acid polymerase and NDPK in aconcentration sufficient to catalyze the production of ATP when in thepresence of a nucleic acid, pyrophosphate and ADP.

Preferably, the polymerase is provided in a concentration of about 0.1to 100 U/reaction (i.e., where “U” is units) most preferably at about0.5 U/reaction. Preferably, the NDPK is provided in a concentration of0.1 to 100 U/reaction, most preferably at about 0.1 U/reaction. Infurther preferred embodiments, the mixture is substantially free ofcontaminating ATP.

Similarly, the PRPP synthetase and NDPK reactions can be carried out ina single pot reaction in the PRPP synthetase buffer. Again, in theseembodiments, NDPK activity is sufficient even though conditions for NDPKactivity are suboptimal.

The nuclease-digested sample containing free NMPs and dNMPs can be addedto a reaction mix initially containing PRPP synthetase and NDPK, oradded to a PRPP synthetase reaction followed by addition to a reactionmix containing NDPK. By way of example, certain preferred buffers andreaction components can be found in the Examples. However, it is notintended that the present invention be limited to specific buffers orreaction components.

PRPP synthetase and NDPK can be provided in the same vessel or mixturefor use in the reactions described above. The mixture preferablycontains the PRPP synthetase and NDPK in a concentration sufficient tocatalyze the production of ATP when in the presence of PRPP and ADP.Preferably, the NDPK is provided in a concentration of 0.1 to 100U/reaction, most preferably at about 0.1 U/reaction. Preferably, thePRPP synthetase is provided in a concentration of 0.001 to 10U/reaction, most preferably at about 0.01 U/reaction. If amplificationis desired, the PRPP synthetase reaction is preferably heat inactivated,otherwise the PRPP synthetase converts the added AMP to ATP.

The pyrophosphorolysis reaction and amplification reaction can also beperformed in a single pot reaction. In this single pot reaction, poly(A)polymerase or any suitable template-dependent polymerase can be used,including, but not limited to, AMV reverse transcriptase, MMLV reversetranscriptase, DNA polymerase alpha or beta, Taq polymerase, Tthpolymerase, Tne polymerase, Tne triple mutant polymerase, Tvupolymerase, Ath polymerase, E. coli DNA polymerase I, T4 DNA polymerase,Klenow fragment, Klenow exo minus, or poly(A) polymerase.

In some embodiments, a first enzyme for converting AMP to ADP can bemyokinase (e.g., adenylate kinase) or NMPK, and in other embodiments, asecond enzyme for converting ADP to ATP can be pyruvate kinase or NDPK.In addition, in preferred embodiments, the reaction is fed AMP. Inparticularly preferred embodiments, apyrase-treated AMP is utilized toreduce background due to contaminating ADP and ATP. Preferably 1 μL of 1U/μL apyrase is added to 19 μL of 10 mM AMP, followed by incubation atroom temperature for 30 minutes and heat inactivation of the apyrase byincubation at 70° C. for 10 minutes.

High energy phosphate donors are also added to the reaction. Inpreferred embodiments, when pyruvate kinase is utilized, PEP is added.In other preferred embodiments, when NDPK is utilized, dCTP is added.Preferably, the high energy phosphate donor is added about 15 minutesafter a pre-incubation with the polymerase, although this is notnecessary. These reactions can be characterized as follows:NA_(n)+PP_(i)→NA_(n-1)+XTPXTP+AMP→ADP+XDPADP+D-P→ATP+D  Reaction 7wherein NA is a nucleic acid, XTP is a nucleoside triphosphate (either adeoxynucleoside or ribonucleoside triphosphate), XDP is a nucleosidediphosphate (either a deoxynucleoside or ribonucleoside diphosphate),and D-P is a high energy phosphate donor. It should be appreciated thatthis reaction produces ATP, the preferred substrate for luciferase, fromdNTPs.

The amplification reaction proceeds as described in reaction 7 toproduce a threshold ATP concentration of approximately 1×10⁻¹² Molar in100 μL of sample. Preferably, the polymerase is provided in aconcentration of about 0.1 to 100 U/reaction, most preferably at about0.5 U/reaction. Preferably, the NDPK is provided in a concentration of0.1 to 100 U/reaction, most preferably at about 0.1 U/reaction.Preferably, the mixture is substantially free of contaminating ATP.

Probe-Mediated Specific Nucleic Acid Detection

Depolymerization reactions can be used to interrogate the identity of aspecific base in a nucleic acid. For example, the identity of singlebase point mutations, deletions, or insertions in a nucleic acid can bedetermined as follows.

In one embodiment, a nucleic acid probe is synthesized that issubstantially complementary to a target nucleic acid containing orsuspected of containing a point mutation. It will be recognized thatvarious hybridization conditions can be used, so as to vary thestringency at which hybridization occurs. Thus, depending upon thesystem utilized, the complementarity of the probe can be varied.Depending on the length of the probe, the GC content, and the stringencyof the hybridization conditions, the probe can have as many as 10 basemismatches with the target nucleic acid, and preferably less than 5mismatches. Most preferably, the probe has only one base mismatch withthe target nucleic acid or is completely complementary to the targetnucleic acid.

The nucleic acid probe comprises single-stranded nucleic acid (e.g., DNAor RNA). The probe can be of varying lengths, preferably from about 10to 100 bases, most preferably about 10 to 30 bases. In particularlypreferred embodiments, the probe is complementary to the target at allbases between an interrogation position and 3′ end of the nucleic acidprobe.

In preferred embodiments, the probe is designed to have a predeterminednucleotide at an interrogation position. When the complementary probebase pairs or hybridizes to the target nucleic acid, the base at aninterrogation position aligns with the base in the nucleic acid targetwhose identity is to be determined under conditions such that basepairing can occur. It is contemplated that an interrogation position canbe varied within the probe. For example, in some preferred embodiments,an interrogation position is preferably within 10 bases of the 3′ end ofthe nucleic acid probe. In still other preferred embodiments, aninterrogation position is within 6 bases of the 3′ end of the nucleicacid probe. In particularly preferred embodiments, an interrogationposition is at the next to last or last base at the 3′ end of thenucleic acid probe.

In some preferred embodiments, four different probes of equal length aresynthesized, each having a different nucleotide at an interrogationposition. Accordingly, it is contemplated that in some embodiments, aset of DNA probes includes a first probe with a deoxyadenosine residueat an interrogation position, a second probe with a deoxythymidineresidue at an interrogation position, a third probe with adeoxyguanosine residue at an interrogation position, and a fourth probewith a deoxycytosine residue at an interrogation position. Likewise, itis also contemplated that a set of RNA probes includes a first probewith an adenosine residue at an interrogation position, a second probewith a uridine residue at an interrogation position, a third probe witha guanosine residue at an interrogation position, and a fourth probewith a cytosine residue at an interrogation position.

In the next step of some embodiments, the probe or probes are hybridizedto the target nucleic acid in separate reactions so that a probe nucleicacid-target nucleic acid complex is formed. It is contemplated thathybridization conditions can vary depending on the length and basecomposition of the probe. In the probe-target nucleic acid complex, thenucleotide at an interrogation position is aligned with the specificbase to be identified in the nucleic acid. In embodiments in which a setof probes is utilized, a different reaction is performed with eachprobe. In a multiplex embodiment, the set of probes can be usedsimultaneously. Because the probes differ at an interrogation position,only one of the probes is complementary to the specific base in thetarget nucleic acid which is aligned with an interrogation position.

In the next step of some embodiments, the nucleic acid probe-targetnucleic acid complexes are individually reacted under conditionspermitting depolymerization of the probe. The preferred reactionconditions for depolymerization are described above and in the followingExamples. The nucleotides are then detected. In preferred embodiments,the reaction mix also contains reagents necessary to catalyze theconversion of XTP to ATP equivalents as described in reaction 4 and inthe following Examples. In some preferred embodiments, the nucleotidesand/or ATP produced by the depolymerization reaction are then detectedby either a luciferase or NADH detection system. Complementarity of thebase at an interrogation position of the nucleic acid probe to thecorresponding base in the nucleic acid target is characterized bydetection of a signal generated from ATP following depolymerization.

In particularly preferred embodiments, the identity of the specific baseis determined by comparing the amount of ATP produced in each reaction.Depolymerization of the probe proceeds from its 3′ end. When the base atan interrogation position is not complementary to the specific base inthe nucleic acid, very little or no ATP is produced, and thus no signalresults. In alternative embodiments, this method can be practiced withfrom one to four probes. It is contemplated that utilizing multipleprobes, (e.g., each with a different base at an interrogation position),may prove unnecessary if a positive signal is produced (e.g., with thefirst probe tested).

In yet another preferred embodiment, the probe-mediated specific nucleicacid detection method of the present invention can be used to simplyidentify or detect a nucleic acid of interest. For this method, anucleic acid probe (e.g., DNA or RNA) is utilized which is substantiallycomplementary to the target nucleic acid, which can be RNA or DNA. In aparticularly preferred embodiment, the nucleic acid probe is entirelycomplementary to the target nucleic acid. The nucleic acid probecomprises single-stranded nucleic acid (e.g., DNA or RNA). The probe canbe of varying lengths, preferably from about 10 to about 1000 bases,most preferably about 10 to 100 bases. Detection is carried out asdescribed above. The nucleic acid probe-nucleic acid target complex isexposed to conditions permitting depolymerization of the probe, whichresults in the production of XTPs. Detection of the nucleic acid ofinterest is characterized by a difference in the signal generated by theXTPs produced. Preferably, the XTPs are converted to ATP as describedabove and the ATP detected by a luciferase or NADH detection system.

In another embodiment, the presence or absence of a lesion in the targetnucleic acid can be detected. A lesion may either be an insertionmutation or a deletion mutation in the wild-type target nucleic acid.The wild-type target nucleic acid contains a region of complementarity,to which the nucleic acid probe can hybridize. Thus, the region ofcomplementarity in the wild-type target nucleic acid is defined by the5′ and 3′ ends of the nucleic acid probe. When the region ofcomplementarity contains a lesion, the nucleic acid probe may stillhybridize to the target nucleic acid, but the hybridization is onlypartial. Depending on the size and nature of the lesion, either the 5′or 3′ end of the probe may hybridize to the target nucleic acid, or ahybridization structure characterized by the presence of a loop may beformed. In each of these cases, depolymerization will be prevented.Preferably, the nucleic acid probe is designed so that the lesion to bedetected begins about less than ten bases from 3′ end of the probe,preferably less than about 6 bases. The nucleic acid probe comprisessingle-stranded nucleic acid (e.g., DNA or RNA). The probe can be ofvarying lengths, preferably from about 10 to about 1000 bases, mostpreferably about 10 to 100 bases. Detection of a nucleic acid containinga lesion is characterized by the difference of a signal generated fromthe XTP produced. Preferably, the XTPs are converted to ATP as describedabove and the ATP detected by a luciferase or NADH detection system.

It is contemplated that an increase in the signal (analytical output)produced by the probe-mediated specific nucleic acid detection methodsof the invention can be realized by a novel cycling method. In thisembodiment of the invention, two probes are designed that arecomplementary to each other and have a 3′ overhang at each end when theyhybridize to one another. In preferred embodiments, the probes aredesigned so that the 3′ overhang is a single base overhang. Inalternative embodiments, the probes also can hybridize to a targetnucleic acid. In particularly preferred embodiments, a polymerase thatacts from the 3′ end of nucleic acids and does not recognize 3′overhangs is utilized for the depolymerization reaction, such as Klenowexo−.

In preferred embodiments, the first step of the reaction involveshybridization of an excess of one of the probes to the target nucleicacid in the presence of the polymerase and under conditions permittingdepolymerization as described above. In some embodiments, no 3′ overhangexists, and the depolymerase reaction proceeds from the 3′ end of theprobe. In some embodiments, the reaction is terminated by separating theprobe from the target nucleic acid by heating the probe-target nucleicacid complexes. On average, as few as one base is removed from probesthat were bound to the target nucleic acid, and fractions of shortenedprobes are created.

In the second step, an excess of the second probe is added to thereaction. Due to the law of mass action, the shortened probes producedin the first step have a tendency to bind to the newly addedcomplementary probes, whereas the non-shortened probes bind to thetarget nucleic acid. The shortened probes that bind to the complementaryprobes produce a complex with no 3′ overhang on one end, and aredepolymerized. This effectively doubles the amount of substrateavailable for the depolymerization reaction. Steps one and two can berepeated additional times until the desired level of detection isachieved. In an alternative preferred embodiment, the reactions can becoupled with NDPK as described above, to produce ATP equivalents thatare detectable by a luciferase-based or NADH-based assay system.

The ability to interrogate the identity of a specific base in a nucleicacid also permits discrimination between nucleic acids from differentspecies, or even from different alleles. The ability to detect anddiscriminate between nucleic acids of related or unrelated species alsopermits the identification of species contained within a given nucleicacid-containing sample. For example, the method can be used to determinewhich species of several related bacteria are contained within a sample(e.g., clinical samples, environmental samples, or samples fromnon-human animals).

In preferred embodiments of this method, nucleic acids withsubstantially identical sequences from at least two species or allelesare detected. The region of identity (target nucleic acid sequence)contains at least a single nucleotide mismatch between the species oralleles in at least one predetermined position and also contains a 3′end and a 5′ end or the identification of a nucleic acid sequence uniqueto each species to be identified.

Next, in some embodiments, an RNA or DNA probe that is substantiallycomplementary to the region of identity is synthesized. The probe can beof varying lengths, preferably from about 10 to 1000 bases, mostpreferably about 10 to 100 bases. As above, this complementary probeincludes an interrogation position.

An interrogation position can be varied within the probe. For example,an interrogation position is preferably within 10 bases of the 3′ end ofthe nucleic acid probe. More preferably, an interrogation position iswithin 6 bases of the 3′ end of the nucleic acid probe. Most preferably,an interrogation position is at the next to last or last base of the 3′end of the nucleic acid probe.

The nucleic acid probes are designed so that the base at aninterrogation position is complementary to the nucleotide at thepredetermined position of one species or allele, but not another due tothe mismatch. Likewise, a second probe can be synthesized that iscomplementary at an interrogation position to the nucleotide at thepredetermined position of a second species or allele.

This same procedure can be employed to identify the presence or absenceof multiple species within a given sample. In these embodiments, allthat is required is the identification of substantially identicalsequences between species that contain base mismatches or theidentification of a nucleic acid sequence unique to each species to beidentified. Similarly, this procedure can be used for quantitativeanalysis of the number of alleles at a loci in a sample. By comparingthe quantity of analytical output relative to an appropriate internal orexternal control, the number of alleles at a locus can be determined.These comparative quantities can be expressed in terms of the ratio ofone allele to the other in one sample versus that same ratio measurementin a control sample. In this process, events such as loss ofheterozygosity or trisomy can be detected.

For example, a normal heterozygous control has a ratio of about 1:1 withrespect to the two alleles that make up the heterozygote. That is, eachallele of the heterozygote can be detected when a nucleic acid probe isused to detect the presence of that allele. If the quantity ofanalytical output obtained by the release of identifier nucleotide whenthe first and second alleles are detected is expressed as a ratio, therelative amounts of the first and second allele would be about the samefor a sample which is heterozygous at that locus. When a sample has lostheterozygosity, one of the two alleles is not detectable. If the firstallele is lost, then the first allele will not be detected in the sampleif the sample is assayed using a nucleic acid probe for the firstallele. The second allele will, however, be present at a similar amountas would be present in a known heterozygous control sample, so assayingthe sample with a nucleic acid probe for the second allele will providean analytical output. If the quantity of the analytical output for thefirst and second alleles for a sample having a loss of heterozygosity ofthe first allele is expressed as a ratio, the ratio will be about 0:1,indicating the absence of the first allele. Conversely, where the secondallele is lost, the ratio of the quantity of analytical output for thefirst and second allele would be about 1:0, indicating the absence ofthe second allele.

The presence of trisomy of an allele is detected in a similar fashion.In a trisomy event, four outcomes are possible with respect to a firstand second allele. The trisomy can be homozygous for the first allele,in which case three copies of the first allele will be present and nocopies of the second allele will be present. Thus, the ratio of thequantity of analytical output for the first and second allele will be3:0. If the trisomy is homozygous for the second allele, three copies ofthe second allele will be present. The ratio of the quantity ofanalytical output for the first and second allele will be 0:3. Two casesof heterozygous trisomy are possible: two copies of allele one and onecopy of allele two, or one copy of allele one and two copies of alleletwo. These two heterozygous trisomy outcomes can be detected bydetermining the ratios of the quantity of analytical output for thefirst and second alleles, preferably in comparison to a knownheterozygous control sample. If the ratio is 2:1, then the heterozygoustrisomy has two copies of the first allele and one copy of the secondallele. If the ratio is 1:2, then the heterozygous trisomy has one copyof the first allele and two copies of the second allele.

The use of an appropriate control, for example a heterozygous control,allows the appropriate interpretation of the ratios obtained from theanalysis of a sample suspected of having a loss of heterozygosity or oftrisomy.

In the next step of some embodiments, separate reactions are performedutilizing each probe. The probes are hybridized to the target nucleicacid to form a probe nucleic acid-target-nucleic acid complex. In theprobe nucleic acid-target nucleic acid complex, the nucleotide at aninterrogation position of the probe is aligned with the nucleotide atthe predetermined position in the nucleic acid, so that base pairingoccurs. The probe-target nucleic acid complex is then reacted underconditions permitting depolymerization of the probe from its 3′ end.

Preferred conditions for depolymerization (depolymerization conditions)are described herein. The nucleotides are then detected. In somepreferred embodiments, the nucleotides are converted to ATP equivalentsas described in reaction 4 and in the Examples. In preferredembodiments, the ATP is detected by luciferase or NADH detectionsystems.

These embodiments of the present invention permit discrimination betweennucleic acids from different species or alleles, as NTPs are produced bydepolymerization only when the nucleotide at an interrogation positionof the probe is complementary to the nucleotide at the predeterminedposition of the nucleic acid from the species. As described above,significant depolymerization proceeds only if the base at aninterrogation position is complementary to the base at the predeterminedposition in the target nucleic acid. The NTP concentration, includingthe ATP concentration, differs when a mismatch is present as compared towhen a mismatch is not present. These differences can be detected (e.g.,by either an ATP or NADH detection system).

A method contemplated by the present invention has wide applicability inassaying nucleic acids. In some aspects, an endogenous nucleic acid isassayed to determine whether a particular native or mutant sequence ispresent or absent. This type of analysis is sometimes referred to asgenotyping because the genetic makeup of the subject from which thenucleic acid sample is obtained is determined. Speciation, the identityof an organism, such as the identification of a human, dog, chicken,bovine or the like can be determined by use of species-specific nucleicacid probes such as probes to selected regions of the gene encodingcytochrome B.

Using a contemplated method, one can illustratively determine whether ahuman patient, for example, has the Leiden V mutation, a mutant β-globingene, the cystic fibrosis-related gene in the region of the delta 508allele, a mutation in a prothrombin gene, congenital adrenalhyperplasia, a translocation that takes place in the region of the bcrgene along with involvement of a segment of the abl gene, the number ofrepeated sequences in a gene such as are present in THO 1 alleles or theTPOX alleles, as well as the loss of heterozygosity of the locus ofcertain alleles as is found in certain cancers and also allelic trisomy.Genomic typing can also be used to assay plant genomes such as that ofrice, soy or maize, and the genomes of microbes such as Campylobacterjejuni, cytomegalovirus (CMV) or human immunodeficiency virus (HIV) todetermine whether a drug-resistant strain is present in a sample.

A contemplated method can also be utilized to assay for the presence orabsence of nucleic acid that is exogenous to the source of the sample.For example, a contemplated method can be used to assay for the presenceof viruses such as hepatitis C virus (HCV), cytomegalovirus (CMV), humanimmunodeficiency virus (HIV), as well as to determine the viral load inan organism with a disease, such as a human or a plant. A contemplatedmethod can also be used to identify the presence of an exogenous nucleicacid sequence in a plant such as maize, soy or rice. A contemplatedmethod can also be used to assay for the presence of microorganisms suchas Listeria monocytogenes, Campylobacter spp., Salmonella spp., Shigellaspp. or Escherichia coli (including E. coli E0157) in foodstuffs such asmeats, dairy products, and fruit juices.

The determination of an appropriate nucleic acid target sequence usefulfor designing nucleic acid probes for use in a method of the inventionis within the skill of the art. Databases of genetic sequences, such asGenbank, can be used to ascertain the uniqueness of the selected nucleicacid target. Commercially available software for designing PCR primerscan be used to assist in the design of probes for use in the invention.

Determination of Repeated Sequences

A process of the invention is useful for determining the presence ofrepeated sequences in a nucleic acid sample. The repeated known sequencepresent in a nucleic acid target sequence typically has a length of 2 toabout 24 bases per repeat. Di- and tri-nucleotide repeats are well knownin the art. An application of this process is Single Tandem Repeat (STR)detection. Typically, different alleles of the target nucleic acid havedifferent numbers of the repeated sequences, so the determination of thenumber of repeats is useful in genotyping. Such methods have importantapplications in the forensic sciences in identity testing. A method fordetermining the number of repeated known sequences is as follows.

Special nucleic acid probes are designed and obtained that containmultiples of a known repeating sequence. Each probe contains a differentnumber of a repeated sequence that is complementary to that of thenucleic acid target sequence. Each probe has a 5′-terminal lockersequence that is complementary to the non-repeated region of the targetthat is downstream of the repeated region in the target. The probestypically have an identifier nucleotide in the 3′-terminal region, butas described herein, the release of nucleotides from the 3′ terminusduring a depolymerization step of the invention can alternatively beascertained by the size of the remaining probe.

The use of a 5′-terminal locker sequence fixes the 5′-terminus of theprobe relative to the repeated region. Thus, for example when the probehas fewer repeats than the target, it is not free to hybridize anywherethroughout the repeated region, but only along the first matching groupof repeats. in this embodiment, the probe will be completelycomplementary to the target sequence, even though it is shorter than thetarget. However, when the probe has more repeats than the target, theprobe extends into the adjacent non-repeated region and is mismatched atits 3′-terminal region.

In some cases, it is desirable to determine the number of repeats bycomparison with standard nucleic acid samples having known numbers ofrepeats (and how they respond to the various probes). In other cases,the number of repeats can be deduced by the shape of the curve of agraph having its ordinate (x-) axis be the number of repeats in theprobe and its abscissa (y-) axis be the analytical output indicating thenumber of nucleotides released, such as light output in luminescencespectroscopic analysis of all of the released nucleotide converted toATP.

In such a graph, for example when the depolymerizing enzyme is atemplate-dependent polymerase or exoIII, the output is greater when theprobe has the same or fewer repeats than the target, relative to theoutput when the probe has more repeats than the target. The S-shapedcurve changes most rapidly after the number of repeats in the probesurpasses the number of repeats in the target. Thus, the derivative ofthe curve is greatest at that point. Similar results when thedepolymerizing enzyme preferentially releases nucleotides frommismatched substrates, except that the output is less when the probe hasthe same or fewer repeats than the target.

Thus, in a preferred embodiment of a process to determine the number ofrepeats of a known sequence, a plurality of separately treated samplesis provided. Each sample contains a nucleic acid target sequence,containing a plurality of known repeated sequences and a downstreamnon-repeated region on the target relative to the repeated sequences.The sample is hybridized with an above-described nucleic acid probe.

A treated reaction mixture is formed by admixing each treated samplewith a depolymerizing amount of an enzyme whose activity is to releaseone or more nucleotides from the 3′-terminus of a hybridized nucleicacid probe. The treated reaction mixture is maintained for a time periodsufficient to permit the enzyme to depolymerize the hybridized nucleicacid probe and release an identifier nucleotide.

The samples are analyzed for the presence or absence of releasedidentifier nucleotide to obtain an analytical output. The analyticaloutput from the sample whose probe contained the same number of sequencerepeats as present in the target nucleic acid is indicative of anddetermines the number of sequence repeats present in the nucleic acidtarget. The analytical output is obtained by luminescence spectroscopy,mass spectroscopy, fluorescence spectroscopy or absorption spectroscopy,including visualization of the remaining probe, as described herein withregard to the general method of the invention.

In one aspect of the method, the target nucleic acid is homozygous withrespect to the number of the repeated sequences at the two alleles. Inan alternative method of the invention, the target nucleic acid isheterozygous for the repeated sequences.

In one method of the invention, an identifier nucleotide is a nucleotidethat is part of the region containing a repeated sequence. In analternative method of the invention, the nucleic acid probe furthercomprises a second non-repeating sequence that is located downstream ofthe repeated sequences in the probe (3′ of them). This secondnon-repeating sequence is complementary to a non-repeating sequencelocated in the target nucleic acid 5′ to its repeated sequences. In thisembodiment, it is contemplated that an identifier nucleotide of theprobe sequence is part of the region containing the second non-repeatingsequence. Thus, the identifier nucleotide is present in a sequencecontaining 1 to about 20 nucleic acids that is complementary to anon-repeating sequence of the target nucleic acid located in the probe3′ to the repeated sequences.

Extension-Mediated Detection of Known Sequences

One common PCR-based method for determining the presence or absence of aspecific known nucleic acid sequence, such as a mutation (e.g. a geneticpolymorphism), is called an amplification refractory mutation system(ARMS), also known as allele-specific PCR (ASP), PCR amplification ofspecific alleles (PASA) or allele-specific amplification (ASA). In atypical ARMS assay two PCR reactions using different PCR primers areconducted on the same nucleic acid sample. Each of the two PCR primersis designed to have a residue at the 3′-terminus of the primer that iscomplementary to one of the two allelic variants and not to the other.The PCR reaction does not extend from a primer having a 3′-terminalmismatched base, unless the polymerase used has a 3′ to 5′ proofreadingactivity that removes the mismatched base and inserts the correct base.Proofreading repairs the PCR primer and destroys the extensiondiscrimination between the two alleles. Therefore, a polymerase lackinga 3′ to 5′ proofreading activity, such as Taq DNA polymerase, is used insuch an ARMS assay. The extension products are typically ascertainedafter agarose gel electrophoresis with ethidium bromide staining.

In the art, it is known that the discrimination between specificity ofPCR extension from the allele-specific ARMS primers is enhanced by theintroduction of deliberate mismatches near the 3′-terminal nucleotide,although possibly also decreasing overall PCR extension product yield.Other factors known to affect the stability of the hybridization of PCRprimers in an ARMS assay include the position of additional mismatchesin the primer, the GC content of the 5 or 6 nucleotides preceding the 3′nucleotide, and the discriminatory 3′-terminal nucleotide, depending onthe difference between the alleles and the type of mismatch. Thedestabilization is greater when the second mismatch is nearer to the3′-terminal nucleotide. The destabilizing effect of additionalmismatches on ARMS has been ranked qualitatively (CC>CT>GG=AA=AC>GT).

A process of the invention can be used to ascertain matched ormismatched bases at the discriminatory 3′-terminal nucleotides in placeof conducting PCR extension, as demonstrated in Example 58. It iscontemplated that similar factors will affect the stability of hybridsin a process of the invention as has been noted with ARMS, sopreferably, such considerations are taken into effect when designingprobes for use in process of the invention.

A contemplated method can be used to determine the presence or absencein a nucleic acid sample of a restriction endonuclease recognitionsequence that cleaves double-stranded DNA leaving a 5′ overhang or ablunt end. The cleavage product is a nucleic acid target that is asubstrate for an enzyme whose activity is to release one or morenucleotides from the 3′-terminus in a process of the invention.

The use of restriction enzymes known at the time of practicing theinvention to leave 5′ overhangs or a blunt end after cleavage arecontemplated for use in a claimed process. Such restriction enzymes arecurrently well known in the art. The enzymes are commercially availablefrom several companies, including Promega Corp. in Madison, Wisconsin. Alist of some such enzymes can be found in Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor LaboratoryPress (Plainview, N.Y.: 1989).

A process of the invention contemplates the detection of a restrictionendonuclease site to detect the presence or absence of a certain nucleicacid within a sequence, such as an SNP. In such a process, the sequenceadjacent downstream of an SNP in a nucleic acid target is used to designa PCR primer for a PCR amplification that is complementary to thedownstream sequence SNP in a first region of the PCR primer. Preferably,the 3′-terminal of the PCR primer is used to determine the presence orabsence of the SNP. Thus, the 3′-terminal residue of the PCR primereither matches or mismatches with the SNP. Preferably, a destabilizingmismatch is incorporated into this PCR primer, as described above, toenhance the specificity of PCR extension from the PCR primer when the3′-terminal residue matches the SNP. A second region of the PCR primerforces the introduction of a restriction endonuclease recognition siteinto the PCR products. The restriction endonuclease recognition sequenceis selected to provide a substrate for a depolymerization reaction ofthe invention. The PCR reaction is conducted and the product purified.The PCR product is treated with the restriction endonuclease to cleaveat its recognition site leaving a restriction endonuclease cleavageproduct containing an identifier nucleotide. A depolymerizing enzyme ofthe invention is admixed with the PCR product either before or afterrestriction endonuclease cleavage. The admixture is analyzed for therelease of identifier nucleotide after maintaining the admixture underdepolymerizing conditions for a time period sufficient fordepolymerization. As noted herein, the identifier nucleotide need not bea nucleotide analog, but it is possible if a nucleotide analog,including a fluorescently labeled nucleotide, were present in theoriginal PCR primer that became the restriction endonuclease recognitionsequence. As discussed herein, the release of identifier nucleotide isascertained by analyzing the released nucleotide or the remaining probe.

Amplification of the Sample Target or a Detection Target

A target nucleic acid sequence is typically amplified prior to use of acontemplated method. However, where a sufficient number of repeatednucleotide sequences are present in the native sample as in the humanAlu sequence or the E. coli rep sequence, amplification is often notneeded prior to carrying out a contemplated method.

Several methods are known in the art to amplify a region of DNA. Theseinclude polymerase chain reaction, ligase chain reaction, repair chainreaction, amplification of transcripts, self-sustained sequencereplication (3SR), ligation activated transcription (LAT), stranddisplacement amplification (SDA) and rolling circle replication. Aclaimed process contemplates prior treatment of a nucleic acid sampleusing any amplification method known in the art at the time ofpracticing a claimed process to detect the presence of a nucleic acidtarget in the sample.

Polymerase chain reaction (PCR) is very widely known in the art. Forexample, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, thedisclosures of which are incorporated herein by reference, describeprocesses to amplify a nucleic acid sample target using PCRamplification primers which hybridize with the sample target.

As the PCR amplification primers are extended, using a DNA polymerase(preferably thermostable), more sample target is made so that moreprimers can be used to repeat the process, thus amplifying the sampletarget sequence. Typically, the reaction conditions are cycled betweenthose conducive to hybridization and nucleic acid polymerization, andthose that result in the denaturation of duplex molecules.

To briefly summarize, in the first step of the reaction, the nucleicacid molecules of the sample are transiently heated, and then cooled, inorder to denature any double stranded molecules that may be present.Amplification and target primers are added to the amplification reactionmixture at an excess concentration relative to the sample target. Whenthe sample is incubated under conditions conducive to hybridization andpolymerization, the amplification primer hybridizes to the nucleic acidof the sample at a position 3′ to the sequence of the desired sampletarget to be amplified. If the nucleic acid molecule of the sample wasinitially double stranded, the target primer will hybridize to thecomplementary strand of the nucleic acid molecule at a position 3′ tothe sequence of the region desired to be amplified that is thecomplement of the sequence whose amplification is desired. Upon additionof a polymerase, the 3′ ends of the amplification and (if the nucleicacid molecule was double stranded) target primers are extended. Theextension of the amplification primer results in the synthesis of a DNAmolecule having the exact sequence of the complement of the desirednucleic acid sample target. Extension of the target primer results inthe synthesis of a DNA molecule having the exact sequence of the desirednucleic acid sample target.

The PCR reaction is capable of exponentially amplifying the desirednucleic acid sequences, with a near doubling of the number of moleculeshaving the desired sequence in each cycle. Thus, by permitting cycles ofhybridization, polymerization, and denaturation, an exponential increasein the concentration of the desired nucleic acid molecule can beachieved. Reviews of the technique include K. Mullis, Cold Spring HarborSymp. Quant. Biol., 51:263-273 (1986); C. R. Newton & A. Graham,Introduction to Biotechniques: PCR, 2^(nd) Ed., Springer-Verlag (N.Y.:1997).

Ligase chain reaction (LCR) is described in European Patent No.0,320,308. In LCR, two adjacent amplification primers, as well as twoothers that are complementary to them, are present in excess in theamplification reaction, which contains DNA ligase. The amplificationprimers hybridize to the complementary sequence in the sample target,such that the adjacent primers are substrates for ligase only whenhybridized to the sample target. Ligase links the two adjacentlyhybridized amplification probes. In a succession of temperature cycles,preferably with use of a thermostable ligase, the linked probes separatefrom the target and can in turn serve as target sequence for otheramplification probes (Barany, Proc. Natl. Acad. Sci., USA, 88:189-1931991). In a contemplated embodiment of the invention, an LCR method isused to amplify a detection target nucleic acid prior to orsimultaneously with hybridization of a detection probe (interrogationprobe) of the invention and depolymerization to release identifiernucleotides.

In another aspect, the nucleic acid of interest is amplified from thecrude nucleic acid sample using a repair chain reaction (RCR) methodprior to detection of the target. In a RCR method of amplification, twooligonucleotide amplification primers that are complementary to theamplification target and two other primers are provided in excess in thepresence of a thermostable DNA ligase and a thermostable DNA polymerase.When a nucleic acid sample target sequence is present, the amplificationprimers hybridize to the nucleic acid sample at either side of theamplification target leaving a gap between the otherwise adjacentlyhybridized primers. The gap is filled in with DNA polymerase and thenligated with DNA ligase to form a complete complementary copy of theamplification target with the primer on either end. By a succession oftemperature cycles, as in PCR and LCR, the extended primers linked tothe amplification primers can in turn serve as target for new primers.International patent application publication number WO 90/01069.

In another contemplated embodiment of the invention, the nucleic acid ofinterest is amplified from the crude nucleic acid sample using anamplification of transcripts (TAS) method prior to detection of thetarget. In a TAS amplification method, such as that described ininternational patent application publication number WO 88/10315, anamplification cycle comprises three stages.

In the first stage, a cDNA is synthesized from RNA in the presence ofreverse transcriptase and a complementary primer also containing an RNApolymerase promoter, such as a phage RNA promoter. Following thermaldenaturation of the RNA/cDNA heteroduplex, the single-stranded cDNA isreplicated by reverse transcriptase in the presence of an antisenseamplification primer. The DNA homoduplex thus obtained during thissecond stage contains a double-stranded promoter to which a phageDNA-dependent RNA polymerase can bind. The third stage then consists oftranscribing RNA molecules (from 30 to 1000 per template) which canserve as template for the synthesis of additional cDNA to continue theamplification cycle. Davis et al., J. Infect. Dis., 162:13-20 (1990).

In another contemplated embodiment of the invention, the nucleic acid ofinterest is amplified from the crude nucleic acid sample using a methodsimilar to TAS, such as self-sustained sequence replication (3SR),nucleic acid sequence-based amplification (NASBA), and single primersequence replication (SPSR), as an amplification method prior todetection of the target using depolymerization according to theinvention. 3SR is described in international patent applicationpublication number WO 90/06995. NASBA is described in European PatentNo. 0,373,960. SPSR is described in U.S. Pat. No. 5,194,370, thedisclosures of which are herein incorporated by reference. These threemethods use RNA- and DNA-dependent DNA polymerases (reversetranscriptase), ribonuclease H (RNase H; Escherichia coli enzyme and/orenzymatic activity associated with reverse transcriptase) andDNA-dependent RNA polymerase (T7 bacteriophage RNA polymerase).

Briefly, at a fixed temperature (37-47° C.), a continuous process ofreverse transcription and transcription reactions are conducted bymethods well-known in the art in order to replicate an RNA target viacDNA. The RNA polymerase binding site (e.g. T7 phage RNA polymerasesite) is introduced by the primer used for the reverse transcriptionstage. The isothermal denaturation of the RNA/cDNA heteroduplex iseffected by specific hydrolysis of the RNA using RNase H activity. Thefree cDNA is replicated from a second oligonucleotide by reversetranscriptase. The resulting DNA/DNA homoduplex is transcribed into RNAby, for example, T7 RNA polymerase. The product RNA can serve as atemplate to repeat the amplification cycle.

In another contemplated embodiment of the invention, the nucleic acid ofinterest is amplified from the crude nucleic acid sample using aligation activated transcription (LAT) method prior to detection of thetarget. LAT is described in U.S. Pat. No. 5,194,370, whose disclosuresare incorporated herein by reference.

Rolling circle replication is described in U.S. Pat. No. 5,854,033, thedisclosures of which are incorporated herein by reference. Rollingcircle replication reporter systems are useful in detecting the presenceof nucleic acid molecules of interest, and in amplifying targetsequences—either of the nucleic acid sequence of interest or of areporter signifying its presence. In a contemplated embodiment of thepresent invention, such amplification and reporter systems are used inconjunction with a hybridization analysis process of the presentinvention.

Several methods of the invention contemplate the determination of thepresence or absence of a predetermined nucleic acid target in a nucleicacid sample using an open circle primer. The predetermined nucleic acidsample target has a 5′ region and a 3′ region and will be considered a“sense” strand for reference purposes.

An open circle first probe has three regions. The open circle probe hasa 5′-terminal region (the first region of the open circle probe) thathybridizes to a 3′ region of a nucleic acid sample target and a3′-terminal region (the third region of the open circle probe) thathybridizes to a 5′ region of a nucleic acid sample target that may beRNA or DNA. The second region is the between the 3′- and 5′-terminalregions. The circular detection probe is DNA that is antisense relativeto the nucleic acid sample target.

The proper hybridization of the 5′- and 3′-terminal regions of the opencircle probe to the predetermined nucleic acid sample target brings the5′- and 3′-terminal ends of the open circle probe into proximity of eachother. The two ends of the open circle probe can be linked togetherusing ligase, or if there are several bases between the two ends aprocess of the invention contemplates that the intervening bases can befilled in using DNA polymerase or an oligo and then linked using ligase.The linkage of the two ends of the open circle probe results in a closedcircular probe that is hybridized to the predetermined nucleic acidsample target. If the predetermined nucleic acid sample target isabsent, then the open circle probe is unaffected by ligase.

An amplification primer hybridizes to a portion of the second region ofthe closed circular probe. In a contemplated amplification step, a DNAchain (sense) is extended from the amplification primer using a DNApolymerase that has displacement activity. An extension product from theamplification primer is complementary to the second region of the closedcircular probe.

If the open circle probe has not been ligated, amplification will onlyproceed through the 5′-terminal region (the first region) of the opencircle probe. DNA complementary to the portion of the open circle primerfrom 3′-terminal region (the third region) through the portions of thesecond region up to the amplification primer hybridization site is notmade unless open circle primer has been ligated to form a closedcircular primer.

With a closed circular probe, DNA complementary to the entire closedcircular probe is replicated, including a detection target sequence thatis complementary to the detection probe (sometimes referred to as aninterrogation probe). Hybridization of the detection probe to thereplicated detection target sequence is ascertained in a process of theinvention by depolymerization of the hybridized nucleic acid to releaseidentifier nucleotides and analyzing for the presence of thoseidentifier nucleotides.

Such processes of the invention using circular probes contemplate threetarget sequences: a predetermined nucleic acid sample target, anamplification target and a detection target. A sequence complementary tothe detection target is typically designed into the open circle probe. Aprocess contemplates that the detection target complement is not in thefirst region of the open circle probe (the 5′-terminal region), but iseither in the second or third region that will only be replicated if theopen circle probe has been ligated to a closed circle primer.

In an alternative contemplated amplification step, an RNA transcriptionorigin is located in the circular probe at a position upstream (in the5′ direction) of the closed circle probe relative to a region that iscomplementary to a detection target. Transcription from this originoccurs in the 5′ to 3′ direction of the transcript, which makes RNAcomplementary to the first region of the open circular transcript andthen stops unless the circle has been ligated. If the circle wereligated to a closed circular probe, then the RNA transcript will runaround the circle and transcribe a region complementary to the detectiontarget. No stop codons should be in the region of the closed circularprimer between the region complementary to the detection target and thetranscription origin.

An embodiment of the invention contemplates a process to determine thepresence or absence of a predetermined single-stranded nucleic acidtarget sequence. Such a process comprises the following steps.

A nucleic acid target sequence can be RNA or DNA. Several methods areknown in the art to obtain single-stranded nucleic acid. For example,the individual strands of double-stranded DNA can be separated bymelting and one strand removed from the composition by binding with abiotin-labeled probe and binding of that hybrid tostreptavidin-containing conjugate, and by other means well-known in theart, some of which are illustrated elsewhere herein. mRNA, on the otherhand, is single-stranded. A preferred process of the invention providesa means to analyze a total mRNA preparation for the presence of aspecific mRNA sequence within that mixture.

A reaction mixture is provided comprising a pair of complementarynucleic acid probes that form 3′-overhangs on both ends of the duplexformed when hybridized with each other. The first of the probes iscomplementary to the nucleic acid target sequence. The second probe thatis complementary to the first probe thus essentially has the samesequence as the nucleic acid target sequence.

A probe can be RNA or DNA, and may include nucleotide analogs.Preferably, a probe is DNA. In the case of the detection of a specificmRNA, the probe is preferably DNA, and the depolymerizing enzyme ispreferably a reverse transcriptase, such as MMLV RT.

In some embodiments of the invention, it is preferable that the3′-terminal region of a probe hybridizes with total complementarity tothe nucleic acid target sequence. However, it is also contemplated toconduct an interrogation of the presence or absence of a particular baseat an interrogation position within the target as described hereinabove.In some embodiments of the invention, it is preferable that the3′-terminal region of a probe hybridizes with partialcomplementarity—even in the case of depolymerization with an enzymewhose maximum activity is to release nucleotides from nucleic acidhybrid having totally complementarity. As shown in Example 30, theintentional introduction of a destabilizing base mismatch at oneposition enhances the discrimination between a match and mismatch atanother position.

The first probe has an identifier nucleotide in its 3′-terminal region.The second probe can also contain an identifier nucleotide in its3′-terminal region. The identifier nucleotide used depends on thedesired method of analysis for released nucleotide, as discussedhereinabove.

A hybrid can form between a first probe and the nucleic acid targetsequence when the nucleic acid target sequence is present in the nucleicacid sample. Typically, a designed probe that can be the same ordifferent from the first probe and is therefore referred to as the thirdprobe can be added to a nucleic acid sample and permitted to anneal tothe designed probe to form a hybrid with the target nucleic acidsequence.

The first reaction mixture also comprises a depolymerizing amount of anenzyme whose activity is to release one or more nucleotides from a3′-terminus of a hybridized nucleic acid. As discussed hereinabove, theparticular enzyme used is based on the substrates to be depolymerizedand the goals of the analysis. The first reaction mixture is similar tothe above-discussed depolymerization reaction mixtures as far aspreferred sample and enzyme concentrations and other reactionconditions.

The first reaction mixture is maintained for a time period sufficient topermit the enzyme to depolymerize hybridized nucleic acid to release anidentifier nucleotide from the 3′-terminal region of the first probe,and form a treated reaction mixture. The general step is discussed ingreater detail hereinabove. In a contemplated process where the originalnucleic acid target is RNA, and the probe is DNA, the effect may benoticed that a DNA/DNA homoduplex is depolymerized at a faster rate orto a greater extent than a DNA/RNA heteroduplex. This effect dependsupon the enzyme and its relative affinity for various substrates.

The treated first reaction mixture is denatured by subjecting thetreated reaction mixture to denaturing conditions and maintaining thetreated reaction mixture for a time period sufficient to denature thenucleic acid hybrids and form a denatured treated reaction mixture. Theprecise conditions required for denaturation are a function of severalfactors as is discussed hereinabove.

Preferably, the reaction will be heated to a temperature of 90-95° C.for 2-5 minutes.

The denatured treated reaction mixture is subjected to annealing(hybridizing) conditions and maintained for a time period sufficient toform a second reaction mixture that comprises hybrids formed between thefirst probe and the 3′-terminal-depolymerized third nucleic acid probe.Because of the 3′-terminal depolymerization of the third probe, thehybrid formed with the first probe have one end that is blunt or thathas a 5′-overhang (i.e., lacks a 3′-overhang on one end of the duplex).The hybrid formed between the first probe and a nucleic acid targetsequence when the nucleic acid target sequence is present in the nucleicacid sample also has a 5′-overhang.

Further depolymerization as before provides a second treated reactionmixture that contains a further quantity of identifier nucleotides inaddition to those provided by the first depolymerization step. Thatfurther quantity of identifier nucleotides can be about twice theoriginal amount so that the total identifier present is aboutthree-times the original amount.

The second treated reaction mixture is analyzed for the presence ofreleased identifier nucleotide to obtain an analytical output. Theanalytical output indicates the presence or absence of thesingle-stranded nucleic acid target sequence.

The first and third probes are preferably the same. Preferably, prior toanalysis of the first reaction mixture to detect released identifiernucleotide, the denaturation, annealing and depolymerization steps arerepeated to further amplify the number of nucleic acid hybrids fromwhich identifier nucleotides are released.

Not wishing to be bound by theory, it is theorized that in an aboveprocess, the first probe (that is complementary to the single-strandedtarget) is depolymerized, either when it is hybridized to the initialsingle stranded nucleic acid target sequence or when it is hybridized toits complementary probe sequence. Thus, the effective concentration oftarget/probe hybrid increases linearly at each progressive cycle ofdepolymerization. Eventually, a first probe can become too short tohybridize effectively as more and more nucleotides are released from its3′-terminus.

A related embodiment of the invention contemplates a process todetermine the presence or absence of a predetermined double-strandednucleic acid target sequence. A process differs from the single-strandtarget process by (i) the presence of a double-stranded nucleic acidtarget sequence and (ii) third and fourth probes hybridized to separatesequences of DNA that results in an exponential rise in the amount ofidentifier nucleotide rather than a linear rise as noted before.

As with the single-strand target method, the first and third probes arepreferably the same. Preferably, prior to analysis of the amplificationreaction mixture to detect released identifier nucleotide, thedenaturation, annealing and depolymerization steps are repeated tofurther amplify the number of nucleic acid hybrids from which identifiernucleotides are released.

It should also be apparent that the single-strand or double strandmethod can also be carried out by addition of the first and secondprobes after one has carried out a depolymerization reaction of atreated reaction mixture and before analysis of identifier nucleotides.Both methods are also particularly useful where a before-mentionedthermostable polymerase is used for depolymerization, as well as where athermostable polymerase and a thermostable NDPK are used in a one-potreaction.

Assays Using Hairpin Structures

Although it is preferred that the probes be constructed to be free ofhairpin structures, assays in which hairpin structures are constructedare also useful. An embodiment of the invention, such as demonstrated inExample 50, contemplates use of a hairpin structure for determining thepresence or absence of a nucleic acid target sequence in a nucleic acidsample with a probe that is hybridized to the target and then modifiedto be able to form a hairpin structure. This embodiment comprises thefollowing steps.

A treated sample is provided that contains a nucleic acid sample thatmay include a nucleic acid target sequence having an interrogationposition. The target sequence, if present in the nucleic acid sample ishybridized with a nucleic acid probe. The probe is comprised of at leasttwo sections. The first section contains the probe 3′-terminal about 10to about 30 nucleotides. These nucleotides are complementary to thetarget strand sequence at positions beginning about 1 to about 30nucleotides downstream of the interrogation position. The second sectionof the probe is located at the 5′-terminal region of the probe andcontains about 10 to about 20 nucleotides of the target sequence. Thissame sequence, therefore, exists in both the target and the probe in thesame 5′ to 3′ orientation. This sequence spans the region in the targetfrom the nucleotide at or just upstream (5′) of the interrogationposition, to the nucleotide just upstream to where the 3′-terminalnucleotide of the probe anneals to the target. An optional third sectionof the probe, from zero to about 50, preferably from zero to about 20,nucleotides in length and comprising a sequence that does not hybridizewith either the first or second section, is located between the firstand second sections of the probe.

The probe of the treated sample is extended in a template-dependentmanner, by admixture with dNTPs and a template-dependent polymerase, atleast through the interrogation position, thereby forming an extendedprobe/target hybrid. In a preferred embodiment, the length of the probeextension is limited by omission from the extension reaction of a DNTPcomplementary to a nucleotide of the target sequence that is presentupstream of the interrogation position and absent between the nucleotidecomplementary to the 3′-end of the interrogation position.

The extended probe/target hybrid is separated from any unreacted dNTPs;i.e., purified at least to the degree needed to use the extended probestrand to determine the presence or absence of the interrogation regionin the sample or the identity of the base at the interrogation position.The extended probe/target hybrid is denatured to separate the strands.The extended probe strand is permitted to form a hairpin structure.

It is preferred that the polymerase enzyme utilized for an extensionreaction be a template-dependent polymerase that is free of activitythat adds a 3′-terminal deoxyadenosine in a template-nonspecific manner.Thus, it is preferred to use other than a polymerase such as Taq for acontemplated extension.

A treated reaction mixture is formed by admixing the hairpinstructure-containing composition with a depolymerizing amount of anenzyme whose activity is to release one or more nucleotides from the3′-terminus of an extended probe hairpin structure. The reaction mixtureis maintained under depolymerizing conditions for a time periodsufficient for the depolymerizing enzyme to release 3′-terminusnucleotides, and then analyzed for the presence of released identifiernucleotides. The analytical output indicates the presence or absence ofthe nucleic acid target sequence. That analytical output can bedetermined as discussed elsewhere herein.

A still further embodiment of the invention, such as that termed REAPER™and demonstrated in Example 89 and FIG. 2, also contemplates use ofhairpin structures in determining the presence or absence of a nucleicacid target sequence, or a specific base within the target sequence, ina nucleic acid sample, and comprises the following steps. A treatedsample is provided that contains a nucleic acid sample that may includea nucleic acid target sequence hybridized with a first nucleic acidprobe strand (FIG. 2A).

The hybrid is termed the first hybrid. The first probe is comprised ofat least two sections. The first section contains the probe 3′-terminalabout 10 to about 30 nucleotides that are complementary to the targetnucleic acid sequence at a position beginning about 5 to about 30nucleotides downstream of the target interrogation position. The secondsection of the first probe contains about 5 to about 30 nucleotides thatare a repeat of the target sequence from the interrogation position toabout 10 to about 30 nucleotides downstream of the interrogationposition, and does not hybridize to the first section of the probe. Thatis, the second sequence is a repeat of the region in the target sequencefrom the interrogation position downstream to the position where the3′-terminal nucleotide of the first probe aligns with the target. Anoptional third section of the probe, located between the first andsecond sections of the probe, is zero to about 50, preferably to about20, nucleotides in length and comprises a sequence that does nothybridize to either the first or second section.

The first hybrid in the treated sample is extended at the 3′-end of thefirst probe, thereby extending the first probe past the interrogationposition and forming an extended first hybrid (FIG. 2B) whose sequenceincludes an interrogation position. The extended first hybrid iscomprised of the original target nucleic acid and extended first probe.The extended first hybrid is then denatured in an aqueous composition toseparate the two nucleic acid strands of the hybridized duplex and forman aqueous solution containing a separated target nucleic acid and aseparated extended first probe.

A second probe, that is about 10 to about 2000, more preferably about 10to about 200, most preferably about 10 to about 30 nucleotides in lengthand is complementary to the extended first probe at a position beginningabout 5 to about 2000, preferably about 5 to about 200, nucleotidesdownstream of the interrogation position in extended first probe, isannealed to the extended first probe, thereby forming the second hybrid(FIG. 2C). The second hybrid is extended at the 3′-end of the secondprobe until that extension reaches the 5′-end of the extended firstprobe, thereby forming a second extended hybrid (FIG. 2D) whose3′-region includes an identifier nucleotide.

It is preferred that the polymerase enzyme utilized for an extensionreaction be a template-dependent polymerase that is free of activitythat adds a 3′-terminal deoxyadenosine in a template-nonspecific manner.Thus, it is preferred to use other than a polymerase such as Taq for acontemplated extension.

An aqueous composition of the extended second hybrid is denatured toseparate the two nucleic acid strands; i.e., the extended second probeand the extended first probe. The aqueous composition so formed iscooled to form a “hairpin structure” from the separated extended secondprobe (FIG. 2E) when the target sequence is present in the originalnucleic acid sample. Thus, when the target sequence is present in theoriginal nucleic acid sample, the 3′-terminal sequence of the secondextended probe in the second extended hybrid hybridizes with thesequence of the second extended probe from a region comprising theinterrogation position and nucleotides downstream from the interrogationposition of second extended probe to the nucleotide position where the3′-terminal nucleotide of the original (first-named) probe annealed tothe original target.

A treated reaction mixture is formed by admixing the hairpinstructure-containing composition with a depolymerizing amount of anenzyme whose activity is to release one or more nucleotides from the3′-terminus of a nucleic acid hybrid. The reaction mixture is maintainedunder depolymerizing conditions for a time period sufficient to release3′-terminal region identifier nucleotides, and then analyzed for thepresence of released identifier nucleotides. The analytical outputindicates the presence or absence of the nucleic acid target sequence.Again, the analytical output can be determined by one of the severalmethods discussed elsewhere herein.

As was the case in the previous embodiment, dNTPs are utilized in theextension reactions. It is preferred that the hairpin structures beseparated from the dNTPs prior to depolymerization to enhance theanalysis for the identifier nucleotide.

Kits

Other embodiments of the invention contemplate a kit for determining thepresence or absence of a predetermined nucleic acid target sequence in anucleic acid sample. Such a kit comprises an enzyme whose activity is torelease one or more nucleotides from the 3′ terminus of a hybridizednucleic acid probe and at least one nucleic acid probe, said nucleicacid probe being complementary to nucleic acid target sequence. The kitoptionally further comprises a nucleoside diphosphate kinase.Preferably, the nucleoside diphosphate kinase is that encoded byPyrococcus furiosis. The kit optionally further comprises instructionsfor detecting said nucleic acid by depolymerization. Preferably theenzyme whose activity is to release nucleotides in the kit is a templatedependent polymerase that, in the presence of pyrophosphate ions,depolymerizes hybridized nucleic acids whose bases in the 3′-terminalregion are matched with total complementarity. Alternatively, the enzymewhose activity is to release nucleotides in the kit exhibits a 3′ to 5′exonuclease activity, depolymerizing hybridized nucleic acids having oneor more mismatched bases at the 3′ terminus of the hybridized probe.

It is to be understood that such a kit is useful for any of the methodsof the present invention. The choice of particular components isdependent upon the particular method the kit is designed to carry out.Additional components can be provided for detection of the analyticaloutput, as measured by the release of identifier nucleotide, or bydetection of the remaining probe after depolymerization. For example,ethidium bromide can be provided in the kits of the invention fordetection of a probe that has had identifier nucleotide released fromthe 3′-terminal region.

The instructions present in such a kit instruct the user on how to usethe components of the kit to perform the various methods of the presentinvention. These instructions can include a description of the detectionmethods of the invention, including detection by luminescencespectroscopy, mass spectrometry, fluorescence spectroscopy, andabsorbance spectroscopy.

In another embodiment, the invention contemplates a kit for determiningthe presence or absence of at least one predetermined nucleic acidtarget sequence in a nucleic acid sample comprising the followingcomponents: an enzyme whose activity in the presence of pyrophosphate isto release identifier nucleotide as a nucleoside triphosphate fromhybridized nucleic acid probe; adenosine 5′ diphosphate; pyrophosphate;a nucleoside diphosphate kinase; and at least one nucleic acid probe,wherein the nucleic acid probe is complementary to the predeterminednucleic acid target sequence.

Preferably, the enzyme whose activity in the presence of pyrophosphateis to release identifier nucleotides is selected from the groupconsisting of the Tne triple mutant DNA polymerase, Klenow exo−, Klenow,T4 DNA polymerase, Ath DNA polymerase, Taq DNA polymerase and Tvu DNApolymerase. Preferably, the nucleoside diphosphate kinase is thatencoded by Pyrococcus furiosis.

The kit optionally comprises instructions for use.

In another embodiment, the invention contemplates a kit for determiningthe presence or absence of a predetermined nucleic acid target sequencein a nucleic acid sample comprising an enzyme whose activity is torelease one or more nucleotides from the 3′ terminus of a hybridizednucleic acid probe and instructions for use. Such a kit optionallycomprises a nucleoside diphosphate kinase. Preferably, the nucleosidediphosphate kinase is that encoded by Pyrococcus furiosis. The kitfurther optionally comprises a nucleic acid probe complementary to thepredetermined nucleic acid target sequence.

In other embodiments of the present invention, nucleic acid detectiontest kits are provided for performing a depolymerization methodcontemplated by this invention, and particularly a depolymerizationdetection method.

In one embodiment, the kit includes a vessel containing an enzymecapable of catalyzing pyrophosphorolysis, including, but not limited toTaq polymerase, Tne polymerase, Tne triple mutant polymerase, Tthpolymerase, Tvu polymerase, Ath polymerase, T4 DNA polymerase, Klenowfragment, Klenow exo minus, E. coli DNA polymerase I, AMV reversetranscriptase, MMLV reverse transcriptase, or poly(A) polymerase. Inanother embodiment, the kit contains a vessel that contains anexonuclease such as S1 nuclease, nuclease BAL 31, mung bean nuclease,exonuclease III and ribonuclease H.

Either of the above enzyme types is utilized in a contemplated method ina depolymerizing effective amount. That is, the enzyme is used in anamount that depolymerizes the hybridized probe to release an identifiernucleotide. This amount can vary with the enzyme used and also with thetemperature at which depolymerization is carried out. An enzyme of a kitis typically present in an amount of about 0.1 to 100 U/reaction; inparticularly preferred embodiments, the concentration is about 0.5U/reaction. An amount of enzyme sufficient to carry out at least oneassay, with its controls is provided.

As noted elsewhere, the preferred analytical output for determining thepresence or absence of identifier nucleotide is luminescence caused bythe reaction of ATP with luciferin in the presence of luciferase. A kitcontaining a pyrophosphorylation enzyme for use in DNA detection usingluminescence also preferably includes a vessel containing NDPK and avessel containing ADP. Similarly, a kit containing an exonuclease enzymefor use in DNA detection using luminescence also preferably includes avessel containing PRPP synthetase and a vessel containing ADP. The NDPKor PRPP synthetase is provided in concentration of about 0.01 to 100U/reaction, preferably about 0.1 to about 1.0 U/reaction.

Preferably, these reagents, and all of the reagents utilized in the kitsdiscussed herein, are free of contaminating ATP and adenylate kinase.Some of the contaminants can be removed from the enzymes by dialysistreatment.

Optionally, the kit contains vessels with reagents for amplification ofdNTPs or NTP to ATP. Amplification reagents include, but are not limitedto pyruvate kinase, adenylate kinase, NMPK, NDPK, AMP (e.g., as theamplification enzymes and substrate), and dCTP or AMP-CPP (e.g., ashigh-energy phosphate donors). In particularly preferred embodiments,the kit can be packaged in a single enclosure including instructions forperforming the assay methods. In some embodiments, the reagents areprovided in containers and are of a strength suitable for direct use oruse after dilution. In alternative preferred embodiments, a standard setcan also be provided in order to permit quantification of results. Inyet other preferred embodiments, test buffers for optimal enzymeactivity are included.

In yet other embodiments, a contemplated kit comprises a nuclease, PRPPsynthetase, PRPP, NDPK, and ADP together with luciferase and luciferin.In preferred embodiments, the nuclease is provided in a concentration ofabout 1 to 500 U/reaction; in particularly preferred embodiments at aconcentration of about 20 U/reaction. In a particularly preferredembodiment, the PRPP synthetase is provided in concentration of about0.01 U/reaction to 10 U/reaction, preferably about 0.1 U/reaction. Insome preferred embodiments, the kit includes all these reagents withluciferase and luciferin being provided as a single reagent solution.

In other preferred embodiments, these reagents include, but are notlimited to, a high energy phosphate donor which cannot be utilized byluciferase, preferably dCTP, and AMP together with luciferase andluciferin. In alternative preferred embodiments, the kit includes allthese reagents with luciferase and luciferin being provided in the samesolution.

In still further embodiments of the present invention, the kitsdescribed above can contain a probe or probes for probe-mediatedspecific nucleic acid detection. In some embodiments, the kit containsat least one nucleic acid probe for a nucleic acid target of interest.In other embodiments, the kits contain multiple probes, each of whichcontain a different base at an interrogation position or which aredesigned to interrogate different target DNA sequences.

In each of the embodiments, the kits contain instructions for use ininterrogating the identity of a specific base within a nucleic acidtarget, for discriminating between two homologous nucleic acid targetsthat differ by one or more base pairs, or for determining whether anucleic acid target contains a deletion or insertion mutation. The typesof nucleic acid probes that can be included in the kits and their usesare described in greater detail below.

EXAMPLE 1 Comparison of Signal Strengths During Allele DeterminationUsing Probes that Interrogate the Same DNA Strand Versus Probes thatInterrogate Different Strands

Because DNA normally exists in a eukaryotic genome as a double-strandedpolymer; in theory, allele discrimination could be performed by:

A) using probes that are essentially identical in sequence (except foran allele discriminating base) and that hybridize to the same DNAstrand; or

B) using two probes that hybridize to different strands of DNA but matchthe sequence of only one allele of the gene at a position where thegenotype is to be determined.

In this example, a comparison is made of the signal strengths of thesetwo types of probes in distinguishing a single nucleotide polymorphism(SNP) target nucleic acid provided as a homozygous target for any knownallele or as a heterozygous target containing different alleles.

Oligonucleotide PH1 (SEQ ID NO:1) is a probe that encodes a segment ofthe human prohibitin gene where SNP exists; it matches the “C” allele.Oligonucleotide PH2 (SEQ ID NO:2) is a probe that encodes the samesegment of the human prohibitin gene but it differs in one base from PH1and matches the “T” allele. These two oligonucleotides hybridize to thesame strand of a target DNA. Oligonucleotide PH4 (SEQ ID NO:3) is aprobe that encodes a segment of the human prohibitin gene where the SNPdefined by PH1 and PH2 exists, however this probe is made to hybridizeto the other target strand than that to which PH1 and PH2 hybridize.

Oligonucleotides PH5 (SEQ ID NO:4) and PH6 (SEQ ID NO:5), when annealedtogether, are a double strand segment of DNA (PH5+6), and each strand islarger than PH1, PH2 or PH4. PH5 and PH6 encode a larger region of thehuman prohibitin gene, and match the “C” allele. This double-strandedtarget was produced by dissolving PH5 and PH6 in water to aconcentration of 1 mg/mL, mixing equal volumes of these solutionstogether, heating the mixture to 95° C. for 5 minutes and then coolingto room temperature over a period of an hour.

Oligonucleotides PH7 (SEQ ID NO:6) and PH8 (SEQ ID NO:7) form a doublestrand DNA segment (PH7+8) identical in sequence to PH5+6 except that itcontains the “T” allele of the gene. This double-stranded DNA wasproduced as described above. An equal mass mixture of thedouble-stranded target nucleic acids produced a mixed sample with equalamounts of the two alleles, as exists for samples heterozygous for thesealleles.

PH1, PH2 and PH4 were dissolved in water to a concentration of 1 mg/mL.PH5+6 and PH7+8 were also diluted to 1 μg/mL in water. Equal volumes ofthe double-stranded DNA target segments were diluted and mixed toproduce a mixture containing equal amounts of both alleles. Thissolution was labeled PH(5+6)+(7+8). The following solutions wereassembled. PH (5 + 6) + Soln PH1 PH2 PH4 PH5 + 6 PH7 + 8 (7 + 8) Water#1 — — — — — — 20 μL #2 1 μL — — — — — 19 μL #3 — 1 μL — — — — 19 μL #4— — 1 μL — — — 19 μL #5 — — — 1 μL — — 19 μL #6 — — — — — 1 μL 19 μL #7— — — — 1 μL — 19 μL #8 1 μL — — 1 μL — — 18 μL #9 — 1 μL — 1 μL — — 18μL #10 1 μL — — — — 1 μL 18 μL #11 — 1 μL — — — 1 μL 18 μL #12 1 μL — —— 1 μL — 18 μL #13 — 1 μL — — 1 μL — 18 μL #14 1 μL — — 1 μL — — 18 μL#15 — — 1 μL 1 μL — — 18 μL #16 1 μL — — — — 1 μL 18 μL #17 — — 1 μL — —1 μL 18 μL #18 1 μL — — — 1 μL — 18 μL #19 — — 1 μL — 1 μL — 18 μL

These solutions were heated to 95° C. for 5 minutes then permitted tocool at room temperature for 10 minutes. The following master mix wasassembled and mixed. Component Amount 10 × DNA Pol Buffer   200 μL(Promega, M195A) Klenow exo− (1 U/μL)  12.5 μL (Promega M218B) 40 mMSodium Pyrophosphate   25 μL (Promega C350B) NDPK (1 U/μL)   10 μL 10 uMADP (Sigma A5285)   20 μL Water 732.5 μL

Twenty microliters of this master mix were added to solutions 1-19 aboveafter cooling at room temperature for 10 minutes and the resultingmixtures were heated at 37° C. for 15 minutes. After this incubation,duplicate 4 μL samples of each of solutions 2-19 and a single 4 μLsample of solution 1 were removed and added to 100 μL L/L reagent(Promega, F202A) and the light produced by the reaction was measuredimmediately using a Turner® TD20/20 luminometer. The following resultswere obtained. Relative light units Solution Reading 1 Reading 2 Avg. #1 5.03  #2 6.83 4.85 5.84  #3 14.0 13.8 13.9  #4 8.52 8.94 8.73  #58.24 8.98 8.61  #6 6.31 6.40 6.36  #7 5.40 5.30 5.35  #8 371.8 472.7422.3  #9 18.8 20.9 19.9 #10 260.9 257.9 259.4 #11 396.5 401.6 399.1 #129.07 9.31 9.19 #13 567.5 536.5 552.0 #14 380.0 408.7 394.4 #15 54.7746.36 50.6 #16 216.7 220.3 218.5 #17 55.43 125.0 90.2 #18 9.25 9.56 9.4#19 114.0 125.0 119.5

The net relative light values for the data above were calculated asfollows. The ratios reported in this example were determined by firstaveraging the results from matching samples, then determining the netlight production from the matching and mismatching samples and dividingthe net light production from the matching reaction by that seen in themismatch reaction. The net light production was determined bysubtracting the estimated light contribution from the probes andtemplate present in the reactions from the total light produced. Thelight production from the template reaction was considered to be thetotal of that contributed from the template specifically and thatcontributed by contaminating ATP from various components. The netincrease from the probes alone was calculated by subtracting the average“No DNA” values from the probe values since this subtracts thecontributions from contaminating ATP from the probe values. Thus, theformula used to determine the net light production from the reactionswas:Net Light=Total light−[(target alone)+(probe alone−No DNA)]

These values were used to determine the signal ratio by dividing thesignal from the “C” allele probe by the signal from the “T” alleleprobe. The calculated values are shown below. Probes Interrogate theProbes Interrogate Same DNA strand Different DNA Strands TemplateGenotype Template Genotype Probe C/C C/T T/T C/C C/T T/T C Probe 412.9252.2 3.6 C Probe 385 211.3 3.8 T Probe 0 388.9 537.7 T Probe 38.3 80.1110.4 Ratio >400 1.54 .006 Ratio 10 2.6 0.034

These data indicate that very different detection ratios are calculatedfrom both sets of probes but that the signal ratios from the differenttarget genotypes are easily distinguished from each other. In addition,the “T” allele probe PH2 gave a relatively low light signal in theabsence of nucleic acid target using the low Klenow exo− additionsemployed in this example. If such manipulations were not used, the lightsignal from the probe alone would be a large contribution to the totalsignal of the samples containing the probe, making sensitive allelediscrimination more difficult. PH1 5′CTGAACATGCCTGCCAAAGACG 3′ SEQ IDNO:1 PH2 5′CTGAACATGCCTGCCAAAGATG 3′ SEQ ID NO:2 PH45′CAGGAACGTAGGTCGGACACAT 3′ SEQ ID NO:3 PH5 5′CTGCTGGGGCTGAACATGCCTGCCAAAGACGTGTCC SEQ ID NO:4GACCTACGTTCCTGGCCCCCTCGAGCT 3′ PH65′CGAGGGGGCCAGGAACGTAGGTCGGACACGTCTTTG SEQ ID NO:5GCAGGCATGTTCAGCCCCAGCAGAGCT 3′ PH75′CTGCTGGGGCTGAACATGCCTGCCAAAGATGTGTCC SEQ ID NO:6GACCTACGTTCCTGGCCCCCTCGAGCT 3′ PH85′CGAGGGGGCCAGGAACGTAGGTCGGACACATCTTTG SEQ ID NO:7GCAGGCATGTTCAGCCCCAGCAGAGCT 3′

EXAMPLE 2 Reduction of Probe-Alone Background Values for Probes Designedto Interrogate a Viral Sequence

In this example, the background light values from probe-alone reactionsare reduced by alteration of reaction conditions. More specifically, thevalues from such background reactions are reduced by lowering the Klenowexo− level in the reactions as shown in Example 43. In addition, theprobes are used to assay the relative probe signal strength values forprobes that hybridize to the same DNA strand versus probes thathybridize to different strands but that interrogate the same nucleotidepolymorphism site.

Oligonucleotides CV11 (SEQ ID NO:8) and CV12 (SEQ ID NO:9) are a pair ofsingle-stranded DNAs that can hybridize together to produce a segment ofthe genome of cytomegalovirus (CMV) in a form sensitive to the druggancyclovir. Oligonucleotides CV13 (SEQ ID NO:10) and CV14 (SEQ IDNO:11) are a pair of single-stranded DNAs that can hybridize together toproduce the same segment of the CMV genome, but differ from CV11 andCV12 in that they contain a SNP that represents a form of the virusresistant to the drug gancyclovir.

Probe oligonucleotide CV15 (SEQ ID NO:12) can hybridize with exacthomology to a segment of CV12. Probe oligonucleotide CV16 (SEQ ID NO:13)is identical to CV15 except that it contains a one base change from theCV15 sequence at the site of the SNP that confers drug resistance to thevirus. Probe oligonucleotide CV17 (SEQ ID NO:14) can hybridize withexact homology to CV11. Probe oligonucleotide CV18 (SEQ ID NO:15) isidentical to CV17 except that it contains a one base change from theCV17 sequence at the site of the SNP that confers drug resistance to thevirus.

The oligonucleotides above were dissolved in water at a concentration of1 mg/mL and the following solutions were assembled. SolutionOligonucleotide Water #1 — 20 μL #2 CV15, 1 μL 19 μL #3 CV16, 1 μL 19 μL#4 CV17, 1 μL 19 μL #5 CV18, 1 μL 19 μL

These solutions were heated at 95° C. for 5 minutes, then cooled at roomtemperature for 10 minutes. A master mix was prepared as in Example 1,containing Klenow exo− at a concentration of 0.25 U/20 μL of solution.Twenty microliters of this solution were added to solutions 1-5 aboveafter they had cooled, and then the resulting mixtures were heated at37° C. for 15 minutes. After this incubation, 4 μL of each solution wereadded to 100 μL of L/L reagent (Promega F202A) and the light productionof the resulting solution was measured immediately using a Turner® TD20/20 luminometer. The following results were obtained. Solution sampledRelative light units #1 13.07 #2 14.98 #3 14.27 #4 28.25 #5 583.70

These results demonstrate that probes CV15-CV17 provide relatively lowprobe-alone light signals at 0.25 U Klenow exo− per reaction but thatprobe CV18-alone provides a very high relative light signal. Thesequence of the CV18 probe can form a hairpin structure such that theterminal 3′ bases hybridize to the sequence 5′TCGTGC 3′ further towardsthe 5′ end of the segment. Although probe CV17 could form the samestructure, the terminal 3′ base of the resulting structure would have amispaired base.

These data exemplify one of the guiding principles of appropriate probedesign for this system: the probes should not be predicted to formstable hairpin structure and, in particular, should not be predicted togive such a structure with the 3′ end of the probe producing a structurethat forms a blunt end or 5′ overhang in the fragment as they may act asa substrate for the depolymerizing enzyme. In addition, the probes usedshould not be predicted to form probe dimer structures with either bluntends or 5′ overhanging ends because such probes can produce highprobe-alone signals in the system and might make them unacceptable foruse.

Due to their low background, probes CV15-CV17 were then selected forfurther study. Equal volumes of oligonucleotides CV11 and CV12 wereannealed together as described in Example 1, as were CV13 and 14. Theannealed solutions of CV11 and CV12, and CV13 and CV14 were labeledCV11+12 and CV13+14, respectively. The following solutions wereassembled. CV(11 + 12) + CV13 (13 + 14) So- CV11 + + Heterozyg lutionCV15 CV16 CV17 12 14 Template Water #1 — — — — — — 20 μL #2 1 μL — — — —— 19 μL #3 — 1 μL — — — — 19 μL #4 — — 1 μL — — — 19 μL #5 — — — 1 μL —— 19 μL #6 — — — — — 1 μL 19 μL #7 — — — — 1 μL — 19 μL #8 1 μL — — 1 μL— — 18 μL #9 — 1 μL — 1 μL — — 18 μL #10 1 μL — — — — 1 μL 18 μL #11 — 1μL — — — 1 μL 18 μL #12 1 μL — — — 1 μL — 18 μL #13 — 1 μL — — 1 μL — 18μL #14 1 μL — — 1 μL — — 18 μL #15 — — 1 μL 1 μL — — 18 μL #16 1 μL — —— — 1 μL 18 μL #17 — — 1 μL — — 1 μL 18 μL #18 1 μL — — — 1 μL — 18 μL#19 — — 1 μL — 1 μL — 18 μL

These solutions were heated at 95° C. for 5 minutes and then permittedto cool for 10 minutes at room temperature. A master mix solution wasassembled as in Example 1 containing Klenow exo− at a finalconcentration of 0.25 U/20 μL. After solutions 1-19 had cooled, 20 μL ofthe master mix solution were added and the resulting solution heated at37° C. for 15 minutes. After this incubation, duplicate 4 μL samples ofsolutions 2-19 and a single sample of solution 1 were taken, added to100 μL of L/L reagent (Promega, F202A) and the light production of themixture measured immediately using a Turner® TD 20/20 luminometer. Thefollowing results were obtained. Relative light units Solution Reading 1Reading 2  #1 10.53 —  #2 11.35 12.16  #3 10.79 12.75  #4 17.70 16.76 #5 12.78 11.12  #6 11.36 11.48  #7 12.38 12.16  #8 348.3 369.3  #973.11 74.48 #10 289.5 283.6 #11 509.8 364.0 #12 120.2 108.6 #13 785.4595.7 #14 764.3 763.3 #15 77.25 73.22 #16 530.9 541.2 #17 476.1 419.6#18 339.4 262.7 #19 943.2 964.0

The results from the readings above were averaged and the net lightunits calculated as described in Example 1. These values were used tocalculate ratios also as described in Example 1. The results of thesecalculations are presented in the tables below, wherein “WT” indicatesthe wild type genotype. Probes Interrogate the Same Probes InterrogateDifferent DNA Strand DNA Strands Template Genotype Template GenotypeProbe C/C C/T T/T C/C C/T T/T WT Probe 345.5 274.0 100.8 WT Probe 745.1518.0 282.1 (CV15) (CV15) Mutant 60.5 424.3 677 Mutant 61.9 435.0 940Probe Probe (CV16) (CV17) Ratio 5.7 1.5 0.15 Ratio 12 1.2 0.33

These data demonstrate that, for this particular SNP, probes that detectthe polymorphism that bind to different strands provide the signal ratioclosest to 1.0 when both nucleic acid targets are present in thereaction (as occurs for samples heterozygous for a particular allele).However either set of probes give clearly different signals dependingupon the genotype of the sample DNA. CV115′CGCTTCTACCACGAATGCTCGCAGACCATGCTGCACGAATACGTCAGAAAG SEQ ID NO:8AACGTGGAGCGTCTGTTGGAGCT 3′ CV125′CCAACAGACGCTCCACGTTCTTTCTGACGTATTCGTGCAGCATGGTCTGCG SEQ ID NO:9AGCATTCGTGGTAGAAGCGAGCT 3′ CV135′CGCTTCTACCACGAATGCTCGCAGATCATGCTGCACGAATACGTCAGAAA SEQ ID NO:10GAACGTGGAGCGTCTGTTGGAGCT 3′ CV145′CCAACAGACGCTCCACGTTCTTTCTGACGTATTCGTGCAGCATGATCTGCG SEQ ID NO:11AGCATTCGTGGTAGAAGCGAGCT 3′ CV15 5′ CTACCACGAATGCTCGCAGAC 3′ SEQ ID NO:12CV16 5′ CTACCACGAATGCTCGCAGAT 3′ SEQ ID NO:13 CV17 5′TGACGTATTCGTGCAGCATGG 3′ SEQ ID NO:14 CV18 5′ TGACGTATTCGTGCAGCATGA 3′SEQ ID NO:15

EXAMPLE 3 Multiplex Analysis of Alleles at One Interrogation Site

For a wide variety of genetic disorders, only a very small percentage ofsamples will have a particular single nucleotide polymorphism (SNP) atany one site. For this reason, it can be much more efficient in thesecases to screen for the presence of groups of mutant alleles and toperform secondary, single probe tests only if there is a positive signalfor any of the probes designed to detect the mutant sites. Such a formof multiplex analysis will be performed in this example.

Multiple probes designed to detect a mutant form of a gene in the CMVgenome are used in one reaction and the signal from this reaction iscompared to that from a probe that is specific for the non-mutatedsequence. In this example, the SNP sites are separated by only one baseand the alleles are provided as pure nucleic acid target species.

Oligonucleotides CV19 (SEQ ID NO:16) and CV20 (SEQ ID NO:17) encode asegment of the CMV genome around position 1784 of the viral genome andthese probes encode the non-mutant form of a gene. Oligonucleotides CV21(SEQ ID NO:18) and CV22 (SEQ ID NO:19) encode the same genome segment asCV19 and CV20 but encode a form of the gene where a Leu codon in theencoded protein is altered to encode a Ser codon.

Oligonucleotides CV23 (SEQ ID NO:20) and CV24 (SEQ ID NO:21) also encodethe same genome segment as CV19 and CV20, but these oligonucleotidesencode a form of the genome where the same Leu codon mutated in CV21 andCV22 is altered to a Phe codon. These oligonucleotides are used here astarget nucleic acids for interrogation in this example.

Oligonucleotide probe CV25 (SEQ ID NO:22) exactly matches a region ofCV19 and is designed to detect the non-mutated form of the gene.Oligonucleotide probe CV26 (SEQ ID NO:23) exactly matches a segmentwithin CV21 and is designed to detect the version of the gene where theLeu codon has been mutated to a Ser codon. Oligonucleotide probe CV27(SEQ ID NO:24) exactly matches a segment within CV24 and is designed todetect the version of the target where the Leu codon has been mutated toa Phe codon.

The target nucleic acid pairs CV19 and CV20, CV21 and CV22, and CV23 andCV24 were dissolved at 1 mg/mL in water, annealed as described inExample 1, and subsequently diluted to 3.3 μg/mL with water. The probesCV25, CV26 and CV27 were dissolved at 1 mg/mL in water.

The following solutions were assembled. CV CV (19 + (21 + CV Solution20) 22) (23 + 24) CV25 CV26 CV27 Water #1 and #2 1 μL — — 1 μL — — 18 μL#3 and #4 1 μL — — — 1 μL 1 μL 17 μL #5 and #6 — 1 μL — 1 μL — — 18 μL#7 and #8 — 1 μL — — 1 μL 1 μL 17 μL #9 and #10 — — 1 μL 1 μL — — 18 μL#11 and #12 — — 1 μL — 1 μL 1 μL 17 μL

These solutions were heated at 95° C. for three minutes then cooled atroom temperature for 10 minutes.

The following master mix was assembled and mixed. Component Volume 10 ×DNA Polymerase Buffer   60 μL (Promega, M195A) 40 mM SodiumPyrophosphate  7.5 μL (Promega, C350B) Klenow exo− (10 U/μL)  7.5 μL(Promega, M218B) NDPK (1 U/μL)   3 μL 10 μM ADP   6 μL Water  216 μL

After solutions 1-12 had cooled at room temperature, 20 μL of thismaster mix were added to each solution, and the solutions were heated to37° C. for 15 minutes. After this heating step, a 4 μL sample of eachsolution was added to 100 μL L/L reagent (Promega, F202A) and the lightproduced by the resulting reaction was read immediately using a Turner®TD 20/20 luminometer. The following results were obtained. Solutionsamples Relative Light Units  #1 115.7  #2 120.9  #3 20.85  #4 20.10  #59.99  #6 9.41  #7 102.4  #8 95.2  #9 12.56 #10 12.54 #11 240.3 #12 238.9

The results from the duplicate solutions were averaged and are presentedin the table below. Average Signal from Probe Types Nucleic Wild TypeMutant Probes Acid Target Probe Multiplexed Ratio* Wild Type 118.3 20.485.78 Target Leu to Ser 9.7 98.8 0.10 Target Leu to Phe 12.6 239.6 0.05Target*Ratio is determined by dividing the signal from the wild type probe bythe signal from the multiplexed mutant probes.

These data show that the use of both mutant probes in one reactionpermits either probe to give a signal if the appropriate target is addedto the reaction. The signal ratios produced by the probes designed todetect the mutant target when either probe matches the target aresignificantly different than from when the wild type target is used withthe wild type probe. Thus, comparison of the signals as described abovepermits the user to know that a mutation is present in the tested targetat either of the interrogation sites. CV195′CTCTTTAAGCACGCCGGCGCGGCCTGCCGCGCGTTGGAGAACGGCAAGCTC SEQ ID NO:16 ACGCA3′ CV20 5′CAGCAGTGCGTGAGCTTGCCGTTCTCCAACGCGCGGCAGGCCGCGCCGGCG SEQ IDNO:17 TGCTT 3′ CV215′CTCTTTAAGCACGCCGGCGCGGCCTGCCGCGCGTCGGAGAACGGCAAGCTC SEQ ID NO:18 ACGCA3′ CV22 5′CAGCAGTGCGTGAGCTTGCCGTTCTCCGCGCGCGGCAGGCCGCGCCGGCGT SEQ IDNO:19 GCTT 3′ CV23 5′CTCTTTAAGCACGCCGGCGCGGCCTGCCGCGCGTTTGAGAACGGCAAGCTCSEQ ID NO:20 ACGCA 3′ CV245′CAGCAGTGCGTGAGCTTGCCGTTCTCAAACGCGCGGCAGGCCGCGCCGGCG SEQ ID NO:21 TGCTT3′ CV25 5′ GGCGCGGCCTGCCGCGCGTTG 3′ SEQ ID NO:22 CV26 5′GGCGCGGCCTGCCGCGCGTCG 3′ SEQ ID NO:23 CV27 5′ GCGTGAGCTTGCCGTTCTCCG 3′SEQ ID NO:24

EXAMPLE 4 Multiplexed Genome Analysis on Multiple Templates

For a wide variety of genetic disorders, only a very small percentage ofsamples exhibit a particular single nucleotide polymorphism at any onesite. For this reason, it can be more efficient in these cases to screenfor the presence of groups of mutant alleles and to perform secondary,single probe tests only if there is a positive signal for any of theprobes designed to detect the mutant sites. Such a form of multiplexanalysis will be performed in this example.

Multiple probes designed to detect a mutant form of two different targetgenes are used in one reaction, and the signal from this reaction iscompared to that from a probe that is specific for one of thenon-mutated sequences. Thus, in this example, multiple SNP sites onmultiple targets are interrogated in one reaction.

The targets and probes used in this study are: FV(1+2) (SEQ ID NO:25 andSEQ ID NO:26, respectively) FV(3+4) (SEQ ID NO:27 and SEQ ID NO:28,respectively), FV5 (SEQ ID NO:29), FV6 (SEQ ID NO:30), 9162 (SEQ IDNO:31), 9165 (SEQ ID NO:32), 9163 (SEQ ID NO:33), 9166 (SEQ ID NO:34),and CV2 (SEQ ID NO:35). A synthetic first nucleic acid target of theFactor V gene was designed to have the wild type sequence that containsa G at position 32 of FV1 (SEQ ID NO:25). The complementary strand, FV2,(SEQ ID NO:26) has 4 additional bases at its 3′ terminus. A secondsynthetic nucleic acid target of Factor V was designed to have theLeiden mutation, an A residue at position 32 of FV3. The mutantcomplementary strand, FV4, also had 4 additional bases at its 3′terminus. The nucleic acid target oligonucleotides, FV1 to FV4, wereseparately dissolved at a concentration of one mg/mL in water.oligonucleotides 9162 and 9163 are complementary and have a segment ofthe wild type CMV genome. Oligonucleotides 9163 and 9166 arecomplementary and have the same segment of the viral genome, but theycontain a single base change present in a known drug resistant form ofthe virus. Equal volumes of one mg/mL 9162 and 9165 were combined toserve as wild type target for CMV. Equal volumes of one mg/mL 9163 and9166 were combined to serve as the mutant target for CMV.Oligonucleotide CV2 represents an oligonucleotide designed to detect thedrug resistant form of the CMV sequence.

All the target DNAs [FV(1+2), FV(3+4), 9162+9165, 9163+9166] werediluted to 0.3 μg/mL with water. The other oligonucleotides weredissolved to 1 mg/mL with water. These compositions were used toassemble the following solutions. 9162 9163 + + FV (1 + FV (3 + Soln FV5FV6 CV2 9165 9166 2) 4) Water 1 — — — — — — 20 μL 2 — 1 μL — — — — — 19μL 3 — — 1 μL — — — — 19 μL 4 — 1 μL 1 μL — — — — 18 μL 5 — — — 1 μL — —— 19 μL 6 — — — — 1 μL — — 19 μL 7 — — — — — 1 μL — 19 μL 8 — — — — — —1 μL 19 μL 9 — — — 1 μL — 1 μL — 18 μL 10 — — — 1 μL — — 1 μL 18 μL 11 —— — — 1 μL 1 μL — 18 μL 12 — — — — 1 μL — 1 μL 18 μL 13 — — — — — — — 20μL 14 1 μL — — 1 μL — 1 μL — 17 μL 15 1 μL — — — 1 μL 1 μL — 17 μL 16 1μL — — 1 μL — — 1 μL 17 μL 17 1 μL — — — 1 μL — 1 μL 17 μL 18 — 1 μL 1μL 1 μL — 1 μL — 16 μL 19 — 1 μL 1 μL — 1 μL 1 μL — 16 μL 20 — 1 μL 1 μL1 μL — — 1 μL 16 μL 21 — 1 μL 1 μL — 1 μL — 1 μL 16 μL

These 21 solutions, in triplicate, were heated to 92° C. for 11 minutes,then cooled approximately 1 hour at room temperature.

The following master mix was assembled and mixed. Component Volume Water1008 μL 10 × DNA Polymerase Buffer  280 μL (Promega, M195A) Klenow exo−(1 U/μL)  35 μL (Promega, M218B) 40 mM Sodium Pyrophosphate  35 μL(Promega, C350B) 10 μM ADP  28 μL NDPK (1 U/μL)  14 μL

After cooling at room temperature, 20 μL of the master mix were added toeach of the 21 solutions, in triplicate, and they were heated at 37° C.for 15 minutes then placed on ice.

Five microliter samples of the solutions were placed in wells of amicrotiter plate such that a 5 μL sample of each solution, intriplicate, was present within each plate and three such plates wereprepared. The plates were placed into a Luminoskan® microtiter platereading luminometer and this instrument was programmed to add 100 μL ofL/L reagent (Promega, F120B) to each well and immediately read the lightproduced by the reaction in the well.

The individual readings for each solution within each plate wereaveraged and these averages are given below. Relative Light UnitsAverage of Target Probe(s) Plate 1 Plate 2 Plate 3 Plates none FV5 5.082.81 2.99 3.63 none FV6 2.85 2.72 3.59 3.05 none CV2 2.91 2.73 2.60 2.75none FV6 and 2.56 2.75 2.68 2.66 CV2 9162 + 9165 none 2.67 2.59 2.502.59 9163 + 9166 none 2.72 2.59 2.51 2.61 FV(1 + 2) none 2.80 2.52 2.552.62 FV(3 + 4) none 2.75 2.41 2.51 2.56 9162 + 9165 + FV none 2.57 2.532.34 2.48 (1 + 2) 9162 + 9165 + FV none 2.54 2.46 2.40 2.47 (3 + 4)9163 + 9166 + FV none 2.40 2.39 2.45 2.41 (1 + 2) 9163 + 9166 + FV none2.48 2.35 2.42 2.42 (3 + 4) none none 2.53 2.34 2.22 2.36 9162 + 9165 +FV FV5 25.61 28.23 24.08 25.97 (1 + 2) 9162 + 9165 + FV FV6 and 4.754.53 4.32 4.53 (1 + 2) CV2 9163 + 9166 + FV FV5 25.36 27.72 28.98 27.35(1 + 2) 9163 + 9166 + FV FV6 and 44.69 41.14 45.29 43.71 (1 + 2) CV29162 + 9165 + FV FV5 3.91 3.93 4.16 4.00 (3 + 4) 9162 + 9165 + FV FV6and 32.23 30.57 36.55 33.12 (3 + 4) CV2 9163 + 9166 + FV FV5 3.54 3.643.52 3.57 (3 + 4) 9163 + 9166 + FV FV6 and 58.61 59.14 71.77 63.17 (3 +4) CV2

The light values for the reactions were adjusted from the averaged platevalues above by subtracting the average No-DNA signal value andtarget-alone averages and probe-alone values from the total light valuemeasured for the various target and probe combinations. Reactionsinvolving combinations of Target/Probe were further corrected bysubtracting the appropriate adjusted probe-alone and target-alone valuesto yield a net light value. The resulting values are shown in the tablebelow. Targets FV5 Probes Mutant Probes WT CMV, WT Factor V 22.22 1.76Mutant CMV, 23.68 41.00 WT Factor V WT CMV, 0.27 30.35 Mutant Factor VMutant CMV, (−.12) 60.46 Mutant Factor V

As in the previous example, a very distinctive signal pattern is seenwith the various target combinations that were studied. This indicatesthat using multiple mutant probes in a multiplex manner can reduce thenumber of reactions needed to determine if a mutant site is presentwithin the sample. These data show for this assay system that when thesignal from the mutant probe reactions approaches or is greater thanthat seen with the corresponding wild type probe, the sample contains atarget with a mutation in at least one of the sites. In addition, if thesignal for the wild type (WT) probe is far lower than that for themultiplexed mutant probes, it is likely that at least the targetinterrogated by the wild type probe is in the mutant form. FV15′CTAATCTGTAAGAGCAGATCCCTGGACAGGCGAGGAATACAGAGGGCAGCA SEQ ID NO:25GACATCGAAGAGCT 3′ FV25′AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTCGCCTGTCCAGGGATCTG SEQ ID NO:26CTCTTACAGATTAGAGCT 3′ FV35′CTAATCTGTAAGAGCAGATCCCTGGACAGGCAAGGAATACAGAGGGCAGCA SEQ ID NO:27GACATCGAAGAGCT 3′ FV45′AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTTGCCTGTCCAGGGATCTG SEQ ID NO:28CTCTTACAGATTAGAGCT 3′ FV5 5′ CTGCTGCCCTCTGTATTCCTCG 3′ SEQ ID NO:29 FV65′ CTGCTGCCCTCTGTATTCCTTG 3′ SEQ ID NO:30 9162 5′CGTGTATGCCACTTTGATATTACACCCATGAACGTG SEQ ID NO:31CTCATCGACGTCAACCCGCACAACGAGCT 3′ 9165 5′CGTTGTGCGGGTTCACGTCGATGAGCACGTTCATGG SEQ ID NO:32GTGTAATATCAAAGTGGCATACACGAGCT 3′ 9163 5′CGTGTATGCCACTTTGATATTACACCCGTGAACGTG SEQ ID NO:33CTCATCGACGTCAACCCGCACAACGAGCT 3′ 9166 5′CGTTGTGCGGGTTCACGTCGATGAGCACGTTCACGG SEQ ID NO:34GTGTAATATCAAAGTGGCATACACGAGCT 3′ CV2 5′ CACTTTGATATTACACCCGTG 3′ SEQ IDNO:35

EXAMPLE 5 Detection of DNA Sequences in the Genome of Listeria Species

This example provides an assay for the presence of DNA sequences presentin the genome of Listeria in a gene known as the iap gene.Oligonucleotides LM1 (SEQ ID NO:36) and LM2 (SEQ ID NO:37) encode asegment of the iap gene and are exactly complementary to each other.Oligonucleotide probe LM3 (SEQ ID NO:38) was designed to hybridizeexactly with a region of target LM2, and probe LM4 (SEQ ID NO:39) wasdesigned to hybridize exactly to target LM1.

Oligonucleotides LM1-LM4 were dissolved in TE buffer (10 mM Tris, 1 mMEDTA, pH8.0) at a concentration of 500 μg/mL and then were diluted25-fold in TE buffer to obtain solutions at a DNA concentration of 20ng/μL. The following solutions were assembled. 1 × TE SolutionOligonucleotides Buffer #1 LM1, 10 μL 10 μL #2 LM2, 10 μL 10 μL #3 LM3,10 μL 10 μL #4 LM4, 10 μL 10 μL #5 LM1, 10 μL; LM3, 10 μL — #6 LM1, 10μL; LM4, 10 μL — #7 LM2, 10 μL; LM3, 10 μL — #8 LM2, 10 μL; LM4, 10 μL —#9 — 20 μL

These solutions were heated at 95° C. for 3 minutes, then permitted tocool at room temperature for 15 minutes.

The following master mix was assembled. Component Volume/reactionNanopure water (Promega AA399) 12.75 μL 10 × DNA Polymerase Buffer(Promega    2 μL M195A) 40 mM Sodium Pyrophosphate (Promega  0.25 μLC113) ADP, 2 μM*    1 μL NDPK, 0.1 U/μL**    1 μL Klenow Exo− 10 U/μL(Promega M218)    1 μL*Made by dissolving Sigma A5285 in water.**Made by dissolving Sigma N0379 in water.

After solutions 1-9 had cooled, 2 μL samples of the solution were addedto 18 μL of the master mix, in triplicate, the resulting solutions weremixed and incubated at 37° C. for 15 minutes. After this incubation, thetubes were placed on ice. Once all the incubations were on ice, 20 μL ofthe contents of the tubes were added to 100 μL of L/L reagent (Promega,F202A) and the light production of the resulting reaction was measuredimmediately using a Turner® TD 20/20 luminometer. The following datawere obtained. Relative light units Solution Target Probe Reading 1Reading 2 Reading 3 Avg. #1 LM1 — 70.3 69.7 69.0 69.7 #2 LM2 — 39.6 40.845.3 41.9 #3 — LM3 12.2 12.4 13.2 12.6 #4 — LM4 16.9 17.3 17.4 17.2 #5LM1 LM3 57.7 76.5 72.7 69.0 #6 LM1 LM4 1814 1815 1761 1797 #7 LM2 LM356.72 61.1 57.59 58.5 #8 LM2 LM4 67.5 72.4 79.3 73.1

These data show that LM4 produces a strong signal in the reaction withLM1 and thus can be used to detect this DNA sequence.

Oligonucleotides LM1 and LM2 were diluted to 2 ng/μL in 1× TE buffer.These materials were also used to create the following solutions intriplicate. Solution LM1 LM2 LM3 LM4 1 × TE #1 5 μL 5 μL — — 10 μL #2 5μL 5 μL 10 μL — — #3 5 μL 5 μL — 10 μL —

These solutions were heated to 95° C. for 10 minutes, then permitted tocool for 15 minutes at room temperature.

A master mix was made as described earlier in this example. Aftercooling at room temperature, 2 μL of each solution were added to an 18μL sample of this master mix, and the resulting solutions were incubatedat 37° C. for 15 minutes. After this incubation, 2 μL of the solutionwere added to 100 μL of L/L reagent (Promega, F202A) and the lightproduced was immediately read using a Turner® TD 20/20 luminometer.

The following results were obtained Relative light units SolutionReading 1 Reading 2 Reading 3 Avg. NLU* #1 754.4 727.8 752.7 745.0 — #2857.4 801.0 852.3 836.9 91.9 #3 1185 1211 1192 1196 451*Net light units (NLU) were calculated by subtracting the no probereaction average (#1) from the specific probe reaction values.

With both DNA template strands present, both probes provide signalsabove background.

The sequences used were as follows: LM1 5′GAAGTAAAACAAACTACACAAGCAACTACACCTGCGCCTAAAG SEQ ID NO:36TAGCAGAAACGAAAGAAACTCCAGTAG 3′ LM2 5′CTACTGGAGTTTCTTTCGTTTCTGCTACTTTAGGCGCAGGT SEQ ID NO:37GTAGTTGCTTGTGTAGTTTGTTTTACTTC 3′ LM3 5′ GCAACTACACCTGCGCCTAAAGTAGCAGAA3′ SEQ ID NO:38 LM4 5′ TTCTGCTACTTTAGGCGCAGGTGTAGTTCG 3′ SEQ ID NO:39

EXAMPLE 6 Detection of Segments of the Listeria hyl Gene

In this example, a method is described for the detection of a segment ofthe hyl gene from Listeria monocyotogenes.

Oligonucleotides LM5 (SEQ ID NO:40) and LM6 (SEQ ID NO:41) annealexactly to create a region of the hyl gene. LM7 (SEQ ID NO:42) and LM8(SEQ ID NO:43) oligonucleotides are used as interrogation probes withLM7 completely complementary to LM6 and LM8 completely complementary toLM5. Oligonucleotides LM5-8 were dissolved in 1× TE buffer at aconcentration of 500 μg/mL and then were diluted 25 fold in TE buffer toobtain solutions at a DNA concentration of 20 ng/μL. The followingsolutions were assembled. Solution Oligonucleotides 1 × TE Buffer #1LM5, 10 μL 10 μL #2 LM6, 10 μL 10 μL #3 LM7, 10 μL 10 μL #4 LM8, 10 μL10 μL #5 LM5, 10 μL; LM7, 10 μL — #6 LM5, 10 μL; LM8, 10 μL — #7 LM6, 10μL; LM7, 10 μL — #8 LM6, 10 μL; LM8, 10 μL — #9 — 20 μL

These solutions were heated at 95° C. for 3 minutes, then permitted tocool at room temperature for 15 minutes.

The following master mix was assembled. Volume/reaction Nanopurewater(Promega AA399) 12.75 μL 10 × DNA Polymerase Buffer(Promega M195)   2 μL 40 mM Sodium Pyrophosphate(Promega C113)  0.25 μL ADP, 2 μM*   1 μL NDPK, 0.1 U/μL**    1 μL Klenow Exo− 10 U/μL (Promega M128)    1μL*Made by dissolving Sigma A5285 in water.**Made by dissolving Sigma N0379 in water.

After solutions 1-9 had cooled, triplicate 2 μL samples of the solutionwere added to 18 μL master mix and the resulting solutions were mixedand incubated at 37° C. for 15 minutes. After this incubation, the tubeswere placed on ice. Once all the incubations were on ice, 20 μL of thecontents of the tubes were added to 100 μL of L/L reagent (PromegaF202A) and the light production of the resulting reaction was measuredimmediately using a Turner® TD 20/20 luminometer. The following datawere obtained. Relative light units Solution Reading 1 Reading 2 Reading3 Avg. Net Ave #1 28.53 29.62 30.0 29.41 — #2 81.30 75.12 74.68 77.03 —#3 19.88 13.12 12.80 15.26 — #4 1326 1273 1216 1271 — #5 37.24 36.4036.77 36.80 3.78 #6 2582 2336 2169 2362 1089 #7 90.74 90.83 90.64 90.649.97 #8 1596 1671 1787 1684 347.6 #9 12.33 11.16 11.48 11.66 —

The above data indicate that at least oligonucleotide LM8 can be used todetect the target gene sequence represented in LM6.

Oligonucleotides LM5 and LM6 were diluted to 2 ng/μL in 1× TE buffer (10mM Tris, 1 mM EDTA, pH 8.0). These materials were also used to createthe following solutions in triplicate. 1 × Solution LM5 LM6 LM7 LM8 TE#1 5 μL 5 μL — — 10 μL #2 5 μL 5 μL 10 μL — — #3 5 μL 5 μL — 10 μL —

These solutions were heated to 95° C. for 10 minutes, and then cooledfor 15 minutes at room temperature.

Then 2 μl of the solutions were added to triplicate 18 μL samples of themaster mix and then the resulting solutions were incubated at 37° C. for15 minutes. After this incubation, 2 μL of the solution were added to100 μL of L/L reagent (Promega, F202A) and the light produced wasimmediately read using a Turner® TD 20/20 luminometer.

The following results were obtained. Relative light units SolutionReading 1 Reading 2 Reading 3 Avg. NLU* #1 442.5 431.8 432.2 435.5 — #2576.1 544.6 580.1 566.9 115.7 #3 1779 1837 1908 1841 1405*Net light units (NLU) determined by subtraction of probe alone values(see table above) and solution #1 values from the average light unitsmeasured.

These results demonstrate that specific detection of the segment of thehyl gene sequence from Listeria can be performed using the componentsdescribed above. Because this gene sequence is specific for Listeria,this indicates that the components can be used for specific detection ofListeria DNA. LM5 5′ CATCGACGGCAACCTCGGAGACTTACGAGATATTTTGAAAAAA SEQ IDNO:40 GGCGCTACTTTTAATCGAGAAACACCA 3′ LM6 5′TGGTGTTTCTCGATTAAAAGTAGCGCCTTTTTTCAAAATATCT SEQ ID NO:41CGTAAGTCTCCGAGGTTGCCGTCGATG 3′ LM7 5′ CTCGGAGACTTACGAGATATTTTGAAAAAA 3′SEQ ID NO:42 LM8 5′ TTTTTTCAAAATATCTCGTAAGTCTCCGAG 3′ SEQ ID NO:43

EXAMPLE 7 Detection of a DNA Sequence from Salmonella

In this example, a method for detection of a gene sequence fromSalmonella is provided.

Oligonucleotides ST1 (SEQ ID NO:44), ST2 (SEQ ID NO:45), ST3 (SEQ IDNO:46), and ST4 (SEQ ID NO:47) were dissolved in 1× TE buffer to 500μg/μL and then were diluted 25 fold in 1× TE buffer to obtain solutionsat a DNA concentration of 20 ng/μL. The following solutions wereprepared. Solution Oligonucleotides 1 × TE Buffer #1 ST1, 10 μL 10 μL #2ST2, 10 μL 10 μL #3 ST3, 10 μL 10 μL #4 ST4, 10 μL 10 μL #5 ST1, 10 μL;ST3, 10 μL — #6 ST1, 10 μL; ST4, 10 μL — #7 ST2, 10 μL; ST3, 10 μL — #8ST2, 10 μL; ST4, 10 μL — #9 — 20 μL

These solutions were heated at 95° C. for 3 minutes, then permitted tocool at room temperature for 15 minutes.

The following master mix was assembled. Component Volume/reactionNanopure water (Promega AA399) 12.75 μL 10 × DNA Polymerase Buffer(Promega    2 μL M195) 40 mM Sodium Pyrophosphate (Promega  0.25 μLC113) ADP, 2 μM*    1 μL NDPK, 0.1 U/μL**    1 μL Klenow Exo− 10 U/μL(Promega M128)    1 μL*Made by dissolving Sigma A5285 in water.**Made by dissolving Sigma N0379 in water.

After solutions 1-9 had cooled, three 2 μL samples of the solution wereadded to 18 μL of the master mix and the resulting solution was mixedand incubated at 37° C. for 15 minutes. After this incubation, the tubeswere placed on ice. Once all the incubations were on ice, 20 μL of thecontents of the tubes were added to 100 μL of L/L reagent, and the lightproduction of the resulting reaction was measured immediately using aTurner® TD 20/20 luminometer. The following data were obtained. Relativelight units Solution Reading 1 Reading 2 Reading 3 Avg. Net Avg. #118.28 18.27 17.97 18.17 — #2 231.9 211.4 226.3 223.2 — #3 11.58 12.5611.34 11.83 — #4 14.00 14.48 14.88 14.45 — #5 21.31 21.20 19.44 20.652.18 #6 3003 2943 2918 2955 2933 #7 2780 2782 2641 2734 2510 #8 256.4269.9 271.1 265.8 39.67 #9 11.63 11.39 11.56 11.52 —

These data indicate that both oligonucleotide probes ST3 and ST4 cangive a very strong specific light signals with single strand target DNAsequence from Salmonella.

Oligonucleotides ST1 and ST2 were diluted to 2 ng/μL in 1× TE buffer (10mM Tris, 1 mM EDTA, pH 8.0). These materials were also used to createthe following solutions in triplicate. Solution ST1 ST2 ST3 ST4 1 × TE#1 5 μL 5 μL — — 10 μl #2 5 μL 5 μL 10 μL — — #3 5 μL 5 μL — 10 μL —

These solutions were heated to 95° C. for 10 minutes, then permitted tocool for 15 minutes at room temperature.

A master mix was made as described earlier in this example. Aftercooling at room temperature, 2 μL of each solution were added to an 18μL sample of this master mix, and then the resulting solutions wereincubated at 37° C. for 15 minutes. After this incubation, 2 μl of thesolution were added to 100 μL of L/L reagent and the light produced wasimmediately read using a Turner® TD 20/20 luminometer.

The following results were obtained. Relative light units SolutionReading 1 Reading 2 Reading 3 Avg. NLU* #1 692.5 728.9 678.3 699.9 — #22448 2389 2311 2382 1683 #3 1742 1778 1738 1752 1053*Net light units (NLU) were determined by subtraction of probe alonevalues (see table above) and solution #1 values from the average lightunits measured.

These data demonstrate that oligonucleotide probes ST3 and ST4 providespecific detection of the DNA target sequence from Salmonella even ifboth DNA strands are present.

Sequences used were as follows: ST1 5′TTTAATTCCGGAGCCTGTGTAATGAAAGAAATCACCGTCACTG SEQ ID NO:44AACCTGCCTTTGTCACC 3′ ST2 5′GGTGACAAAGGCAGGTTCAGTGACGGTGATTTCTTTCATTACACAGGCT SEQ ID NO:45CCGGAATTAAA 3′ ST3 5′ TGTGTAATGAAAGAAATCACCGTCACTGAA 3′ SEQ ID NO:46 ST45′ TTCAGTGACGGTGATTTCTTTCATTACACA 3′ SEQ ID NO:47

EXAMPLE 8 Detection of Poly(A) mRNA Using Reverse Transcriptase and NDPK

This example demonstrates a method for the detection of mRNA,particularly poly(A) mRNA. In this method, an oligo(dT) DNA probe(Promega, C110A) is hybridized to the target mRNA and the hybridizedprobe:target is pyrophosphorylated using a reverse transcriptase andpyrophosphate. As the pyrophosphorylation occurs, the deoxynucleosidetriphosphates are used to convert ADP to ATP using the enzyme NDPK. TheATP of the final solution is then measured using luciferase.

The reactions were assembled as presented in the table below, in whichall volumes are in microliters (μL). The reaction components were:Buffer, 5× MMLV-RT Buffer (Promega, M531A); mRNA, Globin MRNA (Gibco BRLcat# 18103-028 dissolved in H₂O); Poly(dT), 0.2 μM oligo(dT)(50); NaPPi,20 mM Sodium Pyrophosphate, (Promega C113A in deionized water); ADP, 10mM ADP (Sigma A-5285); NDPK, 1 U/μL, (Sigma N-0379); MMLV-RT, (PromegaPart #M531A) 200 U/μL; and 200 U/μL Superscript II (Gibco BRL cat#18064-014).

These reactions were incubated at 37° C. for 30 minutes and 2 μL of thereactions were added to 100 μL of L/L reagent (Promega, F202A). Thelight production of the reactions was immediately measured using aTurner® TD-20e luminometer. The data from these studies are presented inthe data table below. Reaction Components Super- Poly MMLV- Scrip BuffermRNA (dT) NaPPi ADP NDPK RT II H₂O Rx (μL) (μL) (μL) (μL) (μL) (μL) (μL)(μL) μL 1 4 1 of 1 1 2 1 1 — 9 50 ng/μl 2 4 1 of 1 1 2 1 1 — 9 10 ng/μl3 4 1 of 1 1 2 1 1 — 9 2 ng/μl 4 4 1 of 1 1 2 1 1 — 9 400 pg/μl 5 4 1 of1 1 2 1 1 — 9 80 pg/μl 6 4 — 1 1 2 1 1 — 9 7 4 1 of 1 1 2 1 — 1 9 50ng/μl 8 4 1 of 1 1 2 1 — 1 9 10 ng/μl 9 4 1 of 1 1 2 1 — 1 9 2 ng/μl 104 1 of 1 1 2 1 — 1 9 400 pg/μl 11 4 1 of 1 1 2 1 — 1 9 80 pg/μl 12 4 — 11 2 1 — 1 9 DATA TABLE Rx mRNA Light Units 1   5 ng 647.2 2   1 ng 425.43 0.2 ng 113.9 4  40 pg 43.56 5   8 pg 23.66 6 — 21.52 7   5 ng 648.5 8  1 ng 500.4 9 0.2 ng 144.2 10   40 pg 45.85 11    8 pg 28.17 12  —19.71

EXAMPLE 9 Detection of a Specific Message by Use of a DNA Probe ExactlyMatching the Message Sequence and Lack of a Signal when the DNA Probe isMismatched at Its 3′ End

In this Example, a luciferase light signal is generated frompyrophosphorylation of a DNA probe that complements the sequence of atarget RNA species. In addition, evidence is presented to demonstratethat this signal is not generated if the 3′-terminal base of the probedoes not complement the RNA base in the target sequence. These datademonstrate that probe pyrophosphorylation can be used to detect thepresence of specific target RNA sequences and that mutations at specificbases in the target sequence can be detected by use of probes thatshould match the base but that do not give a signal with the message.

A master mix was assembled that contained: Capped Kanamycin RNA (0.62mg/mL)  1.25 μL 5 × MMLV Reaction Buffer    50 μL 40 mM SodiumPyrophosphate   2.5 μL 10 μM ADP   2.5 μL NDPK (1 U/μL)    5 μL MMLV-RT(200 U/μL) (Promega, M1701)  12.5 μL Nanopure water 163.75 μL

Probes one through four were dissolved at a concentration of 1 mg/ml in1× TE buffer.

Probe 1 (SEQ ID NO:48) was designed to exactly complement a segment ofthe coding region of the Kanamycin RNA. Probe 2 (SEQ ID NO:49), Probe 3(SEQ ID NO:50)and Probe 4 (SEQ ID NO:51) were designed to match thesequence of Probe 1 except that the 3′-terminal base of the probe wasaltered to one of each of the other three DNA bases at this position.

Nineteen microliters of the master reaction mix were placed in 10labeled 0.5 mL microfuge tubes and the following additions were made tothe tubes: Tubes 1 and 2, 1 μL 1× TE buffer; Tubes 3 and 4, 1 μL Probe1; Tubes 5 and 6, 1 μL Probe 2; Tubes 7 and 8, 1 μL Probe 3; and Tubes 9and 10, 1 μL Probe 4. The 10 0.5 mL microfuge tubes were incubated at37° C. for 20 minutes to hybridize and form treated samples. Thereafter,2 μL of the contents of the tubes were added to 100 μL L/L reagent(Promega, F202A) and the light output of the reagent was measured usinga luminometer. The following data were collected. Relative LightSolution Units 1 3.989 2 3.458 3 49.95 4 52.24 5 3.779 6 3.727 7 4.394 84.163 9 7.879 10 7.811

These data show that MMLV-RT is able to pyrophosphorylate a DNA probethat hybridized to a target RNA squence and that the free nucleosidetriphosphates that are formed are converted to ATP equivalents that canbe measured using luciferase. In addition, the data show that thissignal is either absent or much weaker (solutions 1,2,5,6,7,8,9,10) whena probe with a 3′ mismatch to the expected base is used in the reaction(compare to tubes 3 and 4). Probe 1 SEQ ID NO: 48 5′GCAACGCTACCTTTGCCATGTTTC 3′ Probe 2 SEQ ID NO: 49 5′GCAACGCTACCTTTGCCATGTTTG 3′ Probe 3 SEQ ID NO: 50 5′GCAACGCTACCTTTGCCATGTTTA 3′ Probe 4 SEQ ID NO: 51 5′GCAACGCTACCTTTGCCATGTTTT 3′

EXAMPLE 10 Detection of a Specific RNA: Globin mRNA

In this Example, the light signal produced from pyrophosphorylation ofDNA probes that are complementary to two regions of globin mRNA iscompared to the signals from two DNA probes that are the exact sequenceof the same regions. Once again, probes that totally complement thetarget RNA are shown to give a signal above background, whereas thosethat do not complement the target RNA give little or no signal.

Probe 5 (SEQ ID NO:52), Probe 6 (SEQ ID NO:53), Probe 7 (SEQ ID NO:54),and Probe 8 (SEQ ID NO:55) were diluted to a concentration of 0.5 mg/mLin 1× TE buffer (10 mM Tris, 1 mM EDTA). Purified globin MRNA (GibcoBRL, 18103-028) as target was dissolved in 1× TE buffer (10 mM Tris, 1mM EDTA) to a concentration of 20 ng/μL.

Hybridization solutions were assembled as follows:

-   Solution 1: 10 μL Probe 5 and 10 μL Globin mRNA-   Solution 2: 10 μL Probe 6 and 10 μL Globin mRNA-   Solution 3: 10 μL Probe 7 and 10 μL Globin MRNA-   Solution 4: 10 μL Probe 8 and 10 μL Globin mRNA-   Solution 5: 10 μL Probe 5 and 10 μL 1× TE buffer-   Solution 6: 10 μL Probe 6 and 10 μL 1× TE buffer-   Solution 7: 10 μL Probe 7 and 10 μL 1× TE buffer-   Solution 8: 10 μL Probe 8 and 10 μL 1× TE buffer-   Solution 9: 10 μL 1× TE buffer, 10 μL Globin mRNA

These solutions were assembled in 0.5 mL tubes, heated to 50° C. for 15minutes and permitted to cool to room temperature for 15 minutes.

The following master reaction mixture was assembled: Nanopure water346.5 μL MMLV-RT 5 × Reaction Buffer (Promega   132 μL M195A) Sodiumpyrophosphate (Promega M531)  16.5 μL NDPK (1 U/μL)   33 μL ADP (2 μM)  33 μL MMLV-RT (adjusted to 100 U/μL)   33 μL (Promega, M1701)

The solution above was mixed and 18 μL placed into 27 tubes. Threetwo-microliter samples of each of the hybridization solutions above wereadded in three of the tubes containing the master reaction mix and thetubes were then incubated at 37° C. for 15 minutes and permitted to coolto room temperature to hybridize and form treated samples. The contentsof the tubes were then added to 100 μL of L/L reagent and the lightproduction of the resulting reaction was measured using a luminometer(Turner® TD20/20). The following results were obtained: HybridizationSolution Light Values Average Probe 5 + RNA 6.555 6.303 6.187 6.348Probe 5 + TE 6.335 5.923 6.046 6.101 Buffer Probe 6 + RNA 137.8 128.5169.2 145.2 Probe 6 + TE 10.24 9.429 9.858 9.842 Buffer Probe 7 + RNA6.235 6.763 6.375 6.458 Probe 7 + TE 6.436 6.545 6.138 6.388 BufferProbe 8 + RNA 90.34 95.42 54.7 80.15 Probe 8 + TE 10.21 12.55 9.37210.71 Buffer TE Buffer + RNA 5.579 6.509 6.388 6.159

These data show that a strong light signal is seen when the reactionmixes containing probes 6 or 8 and target RNA were added to the L/Lreagent but little signal was seen when the probes were incubatedwithout target RNA, or when the target RNA was incubated without theseprobes. In addition, probes 5 and 7 provided very low signals in thepresence or absence of added target RNA. Probes 6 and 8 were designed tocomplement two different regions in the coding region of globin MRNA.Probes 5 and 7 were made to exactly mimic the sequence of these sametarget RNA regions. Thus, these data provide a second example of how thepyrophosphorylation of a probe can be used to detect a specific RNA.Probe 5 SEQ ID NO: 52 5′ ATGGTGCATCTGTCCAGTGAGGAGAA GTCT3′ Probe 6 SEQID NO: 53 5′ AGACTTCTCCTCACTGGACAGATGCA CCAT3′ Probe 7 SEQ ID NO: 54 5′GCTGCTGGTTGTCTACCCATGGACCC 3′ Probe 8 SEQ ID NO: 55 5′GGGTCCATGGGTAGACAACCAGCAGC 3′

EXAMPLE 11 Specific Detection of RNA: Comparison of Signals from RNASpecies that Match Probe Sequences to those from Random Target RNA

To detect specific RNA using the pyrophosphorylation reaction describedin the previous Example it is necessary that the probes not give astrong signal with target RNA species that do not contain the sequenceto be detected. In this Example, the strength of the signal provided byuse of probes designed to detect globin mRNA is compared to the signalseen when these probes are used in reactions with yeast total RNA astarget.

Probe 6 (SEQ ID NO:53), Probe 8 (SEQ ID NO:55) and oligo(dT) (Promega,C110A) were diluted to a concentration of 0.5 mg/mL in 1× TE buffer.Globin mRNA (Gibco BRL, 18103-028) was dissolved in 1× TE buffer to aconcentration of 20 ng/μL. Yeast RNA (Sigma Chemical Co. R3629) wasdissolved in 1× TE buffer to a concentration of 20 ng/μL.

Hybridization solutions were assembled as follows:

10 μL oligo(dT) and 10 μL Globin mRNA

10 μL Probe 6 and 10 μL Globin mRNA

10 μL Probe 8 and 10 μL Globin mRNA

10 μL 1× TE buffer and 10 μL Globin mRNA

10 μL oligo(dT) and 10 μL Yeast RNA

10 μL Probe 6 and 10 μL Yeast RNA

10 μL Probe 8 and 10 μL Yeast RNA

10 μL 1× TE buffer and 10 μL Yeast RNA

These solutions were assembled in 0.5 mL tubes, heated to 50° C. for 15minutes, and then permitted to cool to room temperature for 15 minutesto hybridize and form treated samples.

The following master reaction mixture was assembled: Nanopure water346.5 μL MMLV-RT 5 × Reaction Buffer   132 μL Sodium pyrophosphate(Promega M531)  16.5 μL NDPK (1 U/μL)   33 μL ADP (2 μM)   33 μL MMLV-RT(adjusted to 100 U/μL)   33 μL

The solution above was mixed, and 18 μL were placed into 24 tubes. Threetwo-microliter samples of each of the hybridization solutions above wereadded in three of the tubes containing the master reaction mix and thetubes were incubated at 37° C. for 15 minutes. The contents of the tubeswere then added to 100 μL of L/L reagent and the light production of theresulting reaction was measured using a luminometer (Turner® TD20/20).

The following data were obtained: Hybridization Solution RNA Probe LightUnits Average Globin RNA Oligo(dT) 614.1 680.6 657.7 650.8 Globin RNAProbe 6 93.29 92.19 92.9 92.79 Globin RNA Probe 8 77.13 61.69 69.8969.57 Globin RNA none 4.11 4.07 3.92 4.03 Yeast RNA Oligo(dT) 2.05 2.132.22 2.13 Yeast RNA Probe 6 4.25 4.15 4.46 4.28 Yeast RNA Probe 8 6.214.83 4.37 5.14 Yeast RNA none 1.97 1.53 1.97 1.81

These data show that much higher signals result when the probes areincubated with target globin mRNA than when the probes are incubatedwith yeast total RNA as target. Because the yeast RNA should not containthe globin sequence, the lack of a high signal is expected. The factthat oligo(dT) also provides a low signal suggests that most of thetarget RNA in this preparation is not mRNA, but other forms of RNA.Probe 6 SEQ ID 5′ AGACTTCTCCTCACTGGACAGATGCACC NO: 53 AT3′ Probe 8 SEQID 5′ GGGTCCATGGGTAGACAACCAGCAGC3′ NO: 55

EXAMPLE 12 Specific Detection of RNA: Comparison of Signals from RNASpecies that Match Probe Sequences in Reactions with and without AddedExtraneous Target RNA

For the pyrophosphorylation reaction described in Example 10 to be usedto detect specific target sequences, another requirement of the systemis that the probes should give a very similar signal in the presence andabsence of extraneous RNA. In this Example, the strength of the signalof probes designed to detect target globin MRNA in the presence of alarge amount of yeast RNA is compared to the signal seen in the absenceof added yeast RNA. Hybridization solutions containing various levels ofyeast RNA, Probe 6 (SEQ ID NO:53) or Probe 8 (SEQ ID NO:55) and targetglobin mRNA (Gibco BRL, 18103-028) were assembled by adding 5 μL 500μg/μL either probe 6 or probe 8 to 5 μL 40 ng/μL of target globin mRNAand 10 μL yeast RNA (Sigma Chemical Co. R3629) in 1× TE buffer (10 mMTris, 1 mM EDTA) to produce solutions containing total amounts of yeastRNA of 0, 2, 20, 200, 400, and 800 ng. The solutions were heated at 50°C. for 15 minutes and then permitted to cool to room temperature for 15.

Reaction master mix was assembled as in Example 10 above and 18 μL ofthe mix were placed in 18 tubes. After cooling 15 minutes, 2 μL of thevarious hybridization solutions containing probe 6 were added to thetubes and the tubes were placed in a 37° C. heating block.

After 15 minutes of incubation of the hybridization mixture with thereaction master mix, 20 μL of the solution were added to 100 μL of L/Lreagent (Promega, F202A) and the light output of the resulting reactionwas measured using a Turner® TD-20/20 luminometer.

After the probe 6 data were collected, an identical set of reactions wasperformed using the hybridization solutions containing probe 8.

The following data were obtained:

Probe 6 Reactions

Yeast RNA relative light units Average None 96 109 111 105.3  2 ng 98.485.0 118.5 100.7  20 ng 117.9 110.9 82.7 103.65 200 ng 56.4 110.1 93.286.6 400 ng 115.7 110.7 124.6 117 800 ng 127.6 128.7 143.1 133.1

Probe 8 Reactions

Yeast RNA relative light units Average None 105.8 97.0 82.3 95.0  2 ng84.5 84.6 93.7 87.6  20 ng 99.6 111.7 104.9 105.4 200 ng 83.6 75.9 95.685.1 400 ng 94.7 97.2 81.9 91.2 800 ng 50.7 89.0 82.1 73.9

These data indicate that addition of very large amounts of yeast RNA tothe hybridization reaction does not greatly lower the signal fromhybridized probes for specific target RNA species. Probe 6 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACC NO: 53 AT3′ Probe 8 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCAGC3′ NO: 55

EXAMPLE 13 Mutation Detection Using Probes yo Target Globin mRNA #1:Detection of Mismatched Bases at the 3′ End of the Probe Sequence

The pyrophosphorylation reactions for target RNA detection, such asshown with Probe 6 and Probe 8, require that the probe bepyrophosphorylated by an added polymerase. If the 3′-terminus of theprobe contains a base that does not match the RNA, it might not be asubstrate for the pyrophosphorylation reaction. If this is the case,addition of a probe that detects the presence of a target RNA species toreactions containing a sample that contains the target RNA can indicatethat the RNA is altered in sequence at the base that matches the 3′-endof the probe. Substitution of a new probe that contains thecomplementary base to the altered target RNA sequence then provides thesignal. In this way, the pyrophosphorylation reaction can be used tointerrogate the sequence of RNA species in the region matching the3′-end of the probe.

To test this concept, probes were designed that were identical insequence to Probe 6 (SEQ ID NO:53) and Probe 8 (SEQ ID NO:55), with theexception that the 3′-terminal base of each of these probes was variedto one of each of the other three DNA bases. This Example demonstratesthe use of such a probe set for confirming that the target RNA base atthe 3′-end of the probe matches the expected base by providing a lightsignal after the pyrophosphorylation reaction but that the other probeswith altered 3′ bases do not provide this signal.

The probes 6m1 (SEQ ID NO:56), 6m2 (SEQ ID NO:57) and 6m3 (SEQ ID NO:58)were dissolved in 1× TE buffer to a concentration of 500 ng/μL.

Hybridization solutions containing probe 6, 6m1, 6m2 or probe 6m3, orprobe 8, 8m1 (SEQ ID NO:59), 8m2 (SEQ ID NO:60) or probe 8m3 (SEQ IDNO:61) were assembled by adding 5 μL of 20 ng/μL of target globin mRNA(Gibco BRL) or Tris buffer. The solutions were heated at 50° C. for 15minutes, then permitted to cool to room temperature for 15 minutes tohybridize and form treated samples.

Reaction master mix was assembled as in Example 10 above, and 18 μL ofthe mix were placed in 18 tubes. After cooling for 15 minutes, 2 μL ofthe various hybridization solutions containing probe 6, and probe 6m1through probe 6m3 were added to the tubes and the tubes were placed in a37° C. heating block.

After a 15 minute incubation at 37° C. of the hybridization mixes withthe reaction master mix, 20 μL the reaction were added to 100 μL L/Lreagent (Promega, F202A) and the light output of the reaction measuredimmediately in a Turner® TD20/20 luminometer. The following data wereobtained. RNA Probe (+/−) Light Units Average Probe 6 + 157.3 150 130.5149.9 Probe 6 − 16.2 13.3 11.1 13.6 Probe 6m1 + 7.3 7.4 7.5 7.4 Probe6m1 − 6.8 6.7 6.7 6.7 Probe 6m2 + 7.9 8.8 9.2 8.7 Probe 6m2 − 7.9 7.36.5 7.2 Probe 6m3 + 6.9 7.4 7.4 7.2 Probe 6m3 − 6.1 6.8 7.2 6.7 (noprobe) + 7.0 6.3 7.4 6.9

Reaction master mix was again assembled as in Example 10 above and 18 μLof the mix were placed in 18 tubes. After cooling for 15 minutes, 2 μLof the various hybridization solutions containing probes 8, and probes8m1 through probe 8m3 were added to the tubes and the tubes were placedin a 37° C. heating block.

After a 15 minutes incubation at 37° C. of the hybridization mixes inthe reaction master mix, 20 μL of the reaction were added to 100 μL L/Lreagent (Promega, F202A) and the light output of the reaction measuredimmediately in a Turner® TD20/20 luminometer. The following data wereobtained. RNA Probe (+/−) Light Units Average Probe 8 + 29.1 28.7 25.227.66 Probe 8 − 5.0 4.3 5.9 5.1 Probe 8m1 + 2.5 2.5 2.5 2.5 Probe 8m1 −2.3 2.2 2.4 2.3 Probe 8m2 + 7.4 7.1 5.9 6.8 Probe 8m2 − 2.0 2.1 2.1 2.1Probe 8m3 + 3.4 2.5 2.4 2.8 Probe 8m3 − 2.1 2.1 1.9 2.0 (no probe) + 2.32.2 2.1 2.2

These data again demonstrate that if the 3′ base of a probe is not ableto hybridize to the corresponding base on an RNA target, it will notprovide a strong light signal after the pyrophosphorylation reaction asdescribed above. These data also demonstrate that this method can beused to determine if the terminal base of a probe does complement theexpected base in the target RNA, and thus can be used to confirm thatthe target RNA base at the site of pyrophosphorylation initiation is asexpected. Probe 6 SEQ ID 5′ AGACTTCTCCTCACTGGACAGATGCACC NO: 53 AT3′Probe 8 SEQ ID 5′ GGGTCCATGGGTAGACAACCAGCAGC3′ NO: 55 Probe 6m1 SEQ ID5′ AGACTTCTCCTCACTGGACAGATGCACC NO: 56 AA 3′ Probe 6m2 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACC NO: 57 AG 3′ Probe 6m3 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACC NO: 58 AC 3′ Probe 8m1 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCAGA3′ NO: 59 Probe 8m2 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCAGG3′ NO: 60 Probe 8m3 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCAGT3′ NO: 61

EXAMPLE 14 Mutation Detection Using Probes to Target Globin MRNA #2:Detection of Mismatched Bases Penultimate to the 3′ End of the ProbeSequence

Because Example 13 shows that a mismatch at the 3′ end of a probe can bedetected by the absence of a light signal under conditions permittingpyrophosphorylation, a series of probes corresponding to Probe 6 (SEQ IDNO:53) and Probe 8 (SEQ ID NO:55) were made that had altered bases atthe penultimate base from the 3′ end of the probe sequence.

The probes 6m4, (SEQ ID NO:62), 6m5 (SEQ ID NO:63) and 6m6 (SEQ IDNO:64) were dissolved in 1× TE buffer (10 mM Tris, 1 mM EDTA) to aconcentration of 500 ng/μL.

Hybridization solutions containing probe 6 and probe 6m4 through probe6m6 or probe 8 and probes 8m4 (SEQ ID NO:65), 8m5 (SEQ ID NO:66), and8m6 (SEQ ID NO:67), were assembled by adding 5 μL of 20 ng/μL of globinmRNA (Gibco BRL cat# 18103-028) or Tris-Cl buffer. The solutions wereheated at 50° C. for 15 minutes and then permitted to cool to roomtemperature for 15 minutes to hybridize and form a treated sample.

Reaction master mix was assembled as in Example 10 above and 18 μL ofthe mix were placed in 18 tubes. After cooling for 15 minutes, 2 μL ofthe various hybridization solutions containing probe 6 through probe 6m6were added to the tubes and the tubes were placed in a 37° C. heatingblock.

After a 15 minutes incubation at 37° C. of the hybridization mixes inthe reaction master mix, 20 μL of the reaction were added to 100 μL L/Lreagent (Promega, F202A) and the light output of the reaction measuredimmediately. The following data were obtained. Probe RNA (+/−) LightUnits Average Probe 6 + 138.6 111.6 116.0 122.1 Probe 6 − 14.67 12.289.57 12.17 Probe 6m4 + 7.21 6.82 7.46 7.16 Probe 6m4 − 6.24 5.90 6.286.14 Probe 6m5 + 19.97 19.30 16.80 18.69 Probe 6m5 − 6.27 6.23 6.23 6.23Probe 6m6 + 8.22 6.92 7.02 7.39 Probe 6m6 − 6.40 6.32 5.98 6.23 (noprobe) + 4.91 7.59 5.14 6.24

Reaction master mix was assembled as in Example 10 and 18 μl of the mixwere placed in 18 tubes. After cooling 15 minutes, 2 μL of the varioushybridization solutions containing probe 8 through probe 8m6 were addedto the tubes and the tubes were placed in a 37° C. heating block.

After a 15 minute incubation at 37° C. of the hybridization mixes in themaster mix, 20 μL of the reaction were added to 100 μL L/L reagent(Promega F202A) and the light output of the reaction measuredimmediately. The following data were obtained. Data Table Probe RNA(+/−) Light Units Average Probe 8 + 71.24 55.85 76.33 67.81 Probe 8 −12.65 10.15 6.96 9.91 Probe 8m4 + 5.10 5.48 5.31 5.30 Probe 8m4 − 4.765.08 5.04 4.96 Probe 8m5 + 5.60 5.06 5.61 5.42 Probe 8m5 − 2.63 4.424.88 3.98 Probe 8m6 + 5.68 6.13 5.79 5.87 Probe 8m6 − 4.72 4.60 4.844.72 (no probe) + 5.33 4.64 4.18 4.72

These data demonstrate that if the penultimate base to the 3′ end of aprobe is not able to hybridize to the corresponding base on a targetRNA, very little pyrophosphorolysis occurs and a strong signal is notgenerated. These data also demonstrate that this method can be used todetermine if the penultimate base of a probe does complement theexpected base in the RNA and thus can be used to confirm that the RNAbase at the site of the penultimate base of the probe is as expected.Probe 6 SEQ ID 5′ AGACTTCTCCTCACTGGACAGATGCACC NO: 53 AT3′ Probe 8 SEQID 5′ GGGTCCATGGGTAGACAACCAGCAGC3′ NO: 55 Probe 6m4 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACC NO: 62 CC3′ Probe 6m5 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACC NO: 63 GC3′ Probe 6m6 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACC NO: 64 TC3′ Probe 8m4 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCACC3′ NO: 65 Probe 8m5 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCATC3′ NO: 66 Probe 8m6 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCAAC3′ NO: 67

EXAMPLE 15 Effect of Mismatch Location on the Signal Derived fromPyrophosphorylation of a Probe to a Known Target RNA

Because probes that are mismatched at the 3′-terminal base or thepenultimate 3′-base do not give a light signal following incubation inthe pyrophosphorylation reaction conditions given in Examples 13 and 14above, the following study was performed to determine if a mismatchedbase further within the probe sequence could affect the light signalgenerated from pyrophosphorylation reactions.

Probes 6m7 (SEQ ID NO:68), 6m8 (SEQ ID NO:69), 6m9 (SEQ ID NO:70) and6m10 (SEQ ID NO:71), and probes 8m7 (SEQ ID NO:72), 8m8 (SEQ ID NO:73)and 8m9 (SEQ ID NO:74) were dissolved and diluted in water to 1 mg/mL.Globin mRNA (Gibco BRL Product number #18103-010, lot KB6705) wasdissolved in 10 mM Tris-Cl, pH 7.3 buffer at a concentration of 20ng/μL. Ten microliter hybridization reactions were assembled by mixing 5μL of Probes 6 (SEQ ID NO:53), 6m1 (SEQ ID NO:56), 6m6 (SEQ ID NO:64),and 6m7 through 6m10, 8, (SEQ ID NO:55), 8m3 (SEQ ID NO:61), 8m5 (SEQ IDNO:66) and 8m7-9 with 5 μL of target globin mRNA solution. Control mockhybridization solutions were also made by mixing 5 μL of the probeslisted above with 5 μL 10 mM Tris-Cl pH 7.3 and an RNA alone controlmade by mixing 5 μL target globin mRNA solution with 10 mM Tris-Cl pH7.3. All of these solutions were heated at 50° C. for 15 minutes, andthen were permitted to cool to room temperature for 15 minutes tohybridize and form treated samples.

A master reaction mix was made that contained the following per reactionassembled: Nanopure water 10.5 μL 5 × MMLV-RT Buffer  4.0 μL 40 mMSodium Pyrophosphate  0.5 μL NDPK (0.1 U/μL)  1.0 μL ADP (2 μM)  1.0 μLMMLV-RT enzyme  1.0 μL

Triplicate reactions were formed for each hybridization solution, probecontrol solution and target globin RNA solution. Each of these solutionswas formed by adding two microliters of each solution to 18 μl of masterreaction mix, mixing and incubating the resulting solution at 37° C. for20 minutes. After this incubation, each solution was added to 100 μL ofL/L reagent (Promega, F202A) and the light output of the solution soformed was read immediately using a Turner® TD20/20 luminometer.

The following data were obtained: Mismatch Hybridization Location FromLight Net Solution Probe 3′ End Values Average Average Probe 6 + Globinnone 284.9, 283, 289.3 268.7 mRNA 300.0 Probe 6 − Globin none 20.5, 20.720.7 mRNA 20.8 Probe 6m1 + Globin 1 (terminal) 6.5, 6.3 6.4 1.3 mRNA 6.3Probe 6m1 − Globin 1 (terminal) 5.2, 5.1 5.1 mRNA 5.1 Probe 6m6 + Globin2 10.7, 11.5 11.6 6.6 mRNA 12.7 Probe 6m6 − Globin 2 5.2, 4.9 5.0 mRNA4.8 Probe 6m7 + Globin 3 33.3, 30.5 31.7 27.0 mRNA 31.3 Probe 6m7 −Globin 3 4.4, 4.9 4.7 mRNA 4.7 Probe 6m8 + Globin 4 38.7, 37.7 37.8 33.0mRNA 37.1 Probe 6m8 − Globin 4 4.9, 4.8 4.8 mRNA 4.8 Probe 6m9 + Globin5 68.3, 66.1 67.1 62.1 mRNA 66.8 Probe 6m9 − Globin 5 5.0, 4.9 5.0 mRNA5.1 Probe 6m10 + Globin 6 37.9, 35.6 36.5 31.6 mRNA 36.0 Probe 6m10 −Globin 6 4.9, 4.9 4.9 mRNA 5.0 Probe 8 + Globin none 144.1, 156.3 122.5mRNA 159.0, 165.9 Probe 8 − Globin none 33.7, 33.6 33.8 mRNA 34.1 Probe8m3 + Globin 1 (terminal) 6.2, 6.3 6.2 1.0 mRNA 6.2 Probe 8m3 − Globin 1(terminal) 5.3, 5.1 5.2 mRNA 5.1 Probe 8m5 + Globin 2 6.4, 6.2 6.3 1.1mRNA 6.2 Probe 8m5 − Globin 2 4.9, 4.8 5.2 mRNA 6.0 Probe 8m7 + Globin 38.3, 8.2 8.0 3.1 mRNA 7.6 Probe 8m7 − Globin 3 4.9, 4.9 4.9 mRNA 5.0Probe 8m8 + Globin 4 27.12, 26.4 26.7 21.9 mRNA 26.5 Probe 8m8 − Globin4 4.9, 4.7 4.8 mRNA 4.7 Probe 8m9 + Globin 5 42.5, 43.7 43.8 −7.3 mRNA45.3 Probe 8m9 − Globin 5 53.9, 50.1 51.1 mRNA 49.4 Globin mRNA na 5.7,5.8 5.7 Alone 5.5 No Probe, No na 5.2, 5.2 5.2 RNA 5.3

These data indicate that even mismatches as far as 6 base pairs from the3′ end of the probe can significantly reduce the light output from probepyrophosphorylation reactions where an RNA target and MMLV-RT are usedin the reaction. Thus, such a reduction can be used to indicate that amutation has taken place in a region of an RNA at least 6 base pairs inlength. Probe 6 SEQ ID 5′ AGACTTCTCCTCACTGGACAGATGCACC NO: 53 AT3′ Probe8 SEQ ID 5′ GGGTCCATGGGTAGACAACCAGCAGC3′ NO: 55 Probe 6m1 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACC NO: 56 AA 3′ Probe 6m6 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACC NO: 64 TC3′ Probe 6m7 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCACT NO: 68 AT 3′ Probe 6m8 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCATC NO: 69 AT 3′ Probe 6m9 SEQ ID 5′AGACTTCTCCTCACTGGACAGATGCTCC NO: 70 AT 3′ Probe SEQ ID 5′AGACTTCTCCTCACTGGACAGATGTACC 6m10 NO: 71 AT 3′ Probe 8m3 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCAGT3′ NO: 61 Probe 8m5 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCATC3′ NO: 66 Probe 8m7 SEQ ID 5′GGGTCCATGGGTAGACAACCAGCTGC3′ NO: 72 Probe 8m8 SEQ ID 5′GGGTCCATGGGTAGACAACCAGTAGC3′ NO: 73 Probe 8m9 SEQ ID 5′GGGTCCATGGGTAGACAACCATCAGC3′ NO: 74

EXAMPLE 16 Detection of Target DNA Using a Probe

This study is designed to demonstrate that specific target DNA sequencescan be detected by denaturing the target DNA in the presence of a shortoligonucleotide probe that encodes a nucleotide sequence that canhybridize to the target DNA, permitting the solution containing thedenatured DNA mixture to cool to form probe:target hybrid and a treatedsample, and performing a pyrophosphorylation reaction on the solutionfollowed by transfer of the terminal phosphate of the nucleosidetriphosphates produced to ADP to form ATP. The ATP produced is measuredusing a luciferase/luciferin reaction.

Two microliters of a 1 mg/mL DNA solution of a plasmid containing thekanamycin resistance gene was incubated with 5 μL buffer K (PromegaCorp), 4 μL of Endonuclease Sph I (10U/μL, Promega Corporation), and 39μL nuclease-free water for 1 hour at 37° C. The solution was thenincubated at 70° C. for 10 minutes to inactivate the endonuclease. Thefinal solution was labeled as Sph I-digested pKAN (40 ng/μL).

The following solutions were assembled:

-   Solutions 1 and 2:    -   2 μL Sph I digested PKAN    -   18 μL nuclease-free water-   Solutions 3 and 4:    -   1 μL 1 mg/mL Probe 1 (SEQ ID NO:48)    -   19 μL nuclease-free water-   Solutions 5 and 6:    -   2 μL Sph I digested pKAN    -   1 μL 1 mg/mL Probe 1    -   17 μL nuclease-free water.

These solutions were heated at 95° C. for 3 minutes, and were cooled toroom temperature in approximately 10 minutes by placing them on alaboratory bench to form hybrids and treated samples.

A 2× Master Mix was assembled as follows:

-   40 μL 10× DNA Polymerase buffer (Promega, M195A)-   10 μL 40 mM Sodium Pyrophosphate-   10 μL (10 U/μL) Klenow exo minus DNA Polymerase (Promega, M128B)-   2 μL NDPK at a concentration of 1 U/μL-   4 μL 10 μM ADP-   134 μL nuclease-free water

The Master Mix components were mixed and 20 μL 2× Master Mix were addedto each of the solutions heated to 95° C. after they had cooled to roomtemperature. The reactions were then heated to 37° C. for 20 minutes,and then 4 μL of the reaction were added to 100 μL of L/L reagent(Promega F202A) and the light produced by the reaction was immediatelymeasured using a Turner® 20/20 luminometer. The following data wereobtained. Reaction Light Output #1 5.1 #2 4.6 #3 2.2 #4 2.0 #5 423.4 #6430.5

These results show that a strong light signal can be produced fromreactions containing a target DNA sequence, a probe that hybridizes tothis DNA sequence, and Klenow exo minus. Note that the signal producedis far greater when all the components are present than when either thetarget DNA or probe is not present in the reaction. Probe 1 SEQ ID NO:48 5′ GCAACGCTACCTTTGCCATGTTTC 3′

EXAMPLE 17 Identification of a Target DNA Sequence in Plasmid DNA by Useof Probes that Hybridize to that DNA

The previous Example illustrates that a pyrophosphorylation reaction canbe used to detect specific target DNA sequences with probes thathybridize to the target sequence. Previous examples demonstrate thatsuch a reaction can also be used to detect mutations in RNA sequences ifprobes are designed to identify the base pair present at the 3′ end ofthe probe. This example illustrates an analogous reaction using DNA as atarget that is hybridized with a probe prior to the pyrophosphorylationreaction.

The following solutions were assembled with Probe 1 (SEQ ID NO:48),Probe 2 (SEQ ID NO:49), Probe 3 (SEQ ID NO:50), or Probe 4 (SEQ IDNO:51): pKAN DNA Water* Solution (μL) Probe/μL (μL) 1 and 2 1 — 19 3 and4 1 1 μl Probe 1 18 5 and 6 1 1 μl Probe 2 18 7 and 8 1 1 μl Probe 3 189 and 10 1 1 μl Probe 4 18*Nuclease free water.

These solutions were heated at 95° C. for 3 minutes and cooled to roomtemperature for 10 minutes to form hybrids and a treated sample. A 2×Master Mix was assembled and mixed as described in Example 16, and 20 μLof this Master Mix were added to each of the solutions above. Thesereactions were incubated at 37° C. for 20 minutes, and then 4 μL of eachsolution were added to 100 μL L/L reagent (Promega F202A) and the lightproduction of the resulting reaction was immediately measured using aTurner® 20/20 luminometer. The following data were obtained. ReactionLight Units #1 2.2 #2 2.3 #3 227.5 #4 225.8 #5 28.1 #6 27.1 #7 17.9 #818.3 #9 21.6 #10  21.6

These data demonstrate that probes that exactly match a target DNAsequence present on a plasmid give much higher light signals than doprobes that contain a mismatch at the 3′ end of the probe. Because theprobe can be designed to match the base expected at the site, a drasticdrop in this signal can indicate that the expected base is not presentat the site. This system then can be used to detect mutations in targetDNA that alter a base from an expected sequence to another base. Probe 1SEQ ID NO: 48 5′ GCAACGCTACCTTTGCCATGTTTC 3′ Probe 2 SEQ ID NO :49 5′GCAACGCTACCTTTGCCATGTTTG 3′ Probe 3 SEQ ID NO: 50 5′GCAACGCTACCTTTGCCATGTTTA 3′ Probe 4 SEQ ID NO: 51 5′GCAACGCTACCTTTGCCATGTTTT 3′

EXAMPLE 18 Initial Detection Limit For Plasmid Target DNA by Use ofProbe Pyrophosphorylation

In the previous two examples, plasmid target DNA was specificallydetected using probes that hybridized to a target sequence in the DNA.In this example, a titration of target DNA is carried out in thepyrophosphorylation reaction to determine the level of DNA needed toobtain a signal from this reaction.

The Sph I cut target pKAN DNA (40,000 pg/μL) was serially diluted usingnuclease-free water to obtain concentrations of 10,000, 2,500, 625, 156and 39 pg/μL. Duplicate solutions containing 1 μL each of these DNAtarget solutions, 1 μL Probe 1 (SEQ ID NO:48) and 18 μL nuclease-freewater were assembled as were a pair of solutions containing 1 μL Probe 1and 19 μL of nuclease-free water. All of these solutions were heated at95° C. for 3 minutes and then cooled for 10 minutes to room temperatureto permit hybridization and form a treated sample. A 2× master mix wasmade as described in Example 16 and 20 μL of the mix were then added toall tubes and the tubes incubated at 37° C. for 20 minutes. A samplecontaining 4 μL of the solution was then added to 100 μL of L/L reagent(Promega, F202A) and the light measured using a Turner® 20/20luminometer. The following results were obtained. Data Table ReactionDNA Assayed* Light Units #1 4000 pg 168.4 #2 4000 pg 169.4 #3 1000 pg57.7 #4 1000 pg 77.9 #5   250 pg 19.3 #6   250 pg 21.1 #7  62.5 pg 6.3#8  62.5 pg 6.4 #9  15.6 pg 2.4 #10   15.6 pg 2.3 #11   3.9 pg 1.4 #12  3.9 pg 1.4 #13     0 pg 1.1 #14     0 pg 1.4*This number reflects that relative amount of DNA transferred to L/Lsolution.

These data demonstrate that the detection limit for DNA by this reactionunder these conditions is at least about 62.5 pg of DNA and is morelikely about 15.6 pg of DNA or less. Probe 1 SEQ ID NO: 84 5′GCAACGCTACCTTTGCCATGTTTC 3′

EXAMPLE 19 Detection of β-Galactosidase Target Sequences in Plasmids

In this example, two probes are used that complement each other exactly.One of the probes matches the sequence of the β-galactosidase geneexactly (sense orientation) and the other probe exactly matches thecomplementary strand (antisense orientation) of that gene. This exampledemonstrates that, whereas both probes can be used to detect thepresence of the target β-galactosidase gene in plasmid DNA, the level ofbackground signal given by reactions containing only probe DNA can bevery different.

Probe 23 (SEQ ID NO:75) and Probe 24 (SEQ ID NO:76) were dissolved asdescribed above to a concentration of 500 ng/μL and then diluted innuclease-free water to 100 and 20 ng/μL. Plasmid pGEM7zf+ (Promega) wasdigested with Sac I (Promega) as the target and diluted to give asolution containing 20 ng of plasmid target DNA/μL of solution.

The following solutions were assembled: Plasmid DNA Probe, H₂O Solution(μL) Concentration (μL) #1 1 (none, 1 μL of 1 × TE 18 buffer added) #2 01 μL Probe 23, 500 ng/μL 19 #3 0 1 μL Probe 23, 100 ng/μL 19 #4 0 1 μLProbe 23, 20 ng/μL 19 #5 1 1 μl Probe 23, 500 ng/μL 18 #6 1 1 μL Probe23, 100 ng/μL 18 #7 1 1 μL Probe 23, 20 ng/μL 18 #8 0 1 μL Probe 24, 500ng/μL 19 #9 0 1 μL Probe 24, 100 ng/μL 19 #10  0 1 μL Probe 24, 20 ng/μL19 #11  1 1 μL Probe 24, 500 ng/μL 18 #12  1 1 μL Probe 24, 100 ng/μL 18#13  1 1 μL Probe 24, 20 ng/μL 18

These solutions were heated at 95° C. for 3 minutes, and cooled to roomtemperature to form hybrids and treated samples. Then, 20 μL of 2×Master Mix made as described in Example 16 were added and the solutionsincubated for another 20 minutes at 37° C. Four microliters of eachsolution were then added to 100 μL of L/L reagent (Promega, F202A) andthe light output of the reaction immediately measured using a Turner®TD20/20 luminometer.

The following data were obtained. Reaction Light Output Net LightOutput* #1 2.8 #2 4.0 #3 1.9 #4 1.3 #5 52.4 45.6 #6 13.6 8.9 #7 4.1 0 #834.3 #9 6.6 #10 1.7 #11 59.8 22.7 #12 19.3 9.9 #13 6.0 1.5*Net light output is calculated by subtracting the probe alone and DNAalone values from that obtained with both components present.

These data indicate that both probes can be used to generate a signalindicating the presence of the target region encoding theβ-galactosidase gene matching the probes is present in the plasmid. Theyalso demonstrate that the level of signal produced with a probe in theabsence of target DNA can vary and that the signal from a probe and thecomplement of that probe are not necessarily equal. Probe 23 SEQ ID5′CAGTCACGACGTTGTAAAACGACGGCC NO:75 AGT3′ Probe 24 SEQ ID5′ACTGGCCGTCGTTTTACAACGTCGTGA NO:76 CTG3′

EXAMPLE 20 Detection of Specific Target DNA Sequences on Lambda DNA

In this example, detection of the target β-galactosidase gene in the DNAof a recombinant Lambda phage is demonstrated.

Duplicate solutions were made that contained: Solution 1 and 2, 1 μL 300ng/μL of Lambda gt11 DNA and 19 μL of nuclease-free water; Solution 3and 4, 1 μL 500 ng/μL Probe 23 (SEQ ID NO:75) and 19 μL nuclease-freewater; Solution 5 and 6, 1 μL 300 ng/μL Lambda gt11 DNA, 1 μl 500 ng/μLProbe 23, and 18 μL of nuclease-free water. All of these solutions wereheated at 95° C. for 3 minutes and then cooled to room temperature for10 minutes to permit hybridization to occur between complementarystrands and form treated samples. At this point, 20 μl of 2× master mixmade as described in Example 16 were added and the solutions incubatedfor another 20 minutes at 37° C. A 4 μL sample of eachpyrophosphorolysis reaction was then taken and added to 100 μL of L/Lreagent (Promega, F202A) and the light production of the solutionimmediately measured with a Turner® TD20/20 luminometer. The followingdata were obtained. Reaction DNA Components Light Units #1 Target LambdaDNA 16.5 #2 Target Lambda DNA 7.4 #3 Probe 23 2.9 #4 Probe 23 2.9 #5Target Lambda DNA 88.1 and Probe 23 #6 Target Lambda DNA 70.4 and Probe23

These data indicate that the pyrophosphorylation system can be used todetect a probe hybridized to specific target sequences on lambda gt11DNA. Probe 23 SEQ ID 5′CAGTCACGACGTTGTAAAACGACGGCCAGT3′ NO:75

EXAMPLE 21 Probe Dependent Detection of A PCR Product byPyrophosphorolysis

A 613 bp POR product was synthesized by reverse transcription PCR(RT-PCR) from a 1.2 kb synthetic RNA corresponding to the kanamycinresistance gene in plasmid pKanDeltaCG (Promega). The RNA wassynthesized using a commercial kit from Ambion (MMESSAGE mMACHINE SP6Kit Cat#1340) Austin, Tex. PKanDeltaCG was first linearized with EcoR Ito enable a run-off transcript to be made. The plasmid was digested forone hour at 37° C. in the following reaction:  25 μL 1 mg/mL pKanDeltaCG  10 μL 10 × Multi-Core Buffer (Promega R999A)  5 μL 80 U/μL EcoRI (Promega R6011)  60 μL water 100 μL

Ten microliters 5M NaCl were added to the EcoR I digested DNA and thereaction was extracted with 110 μL phenol:chloroform:isoamyl alcohol(49:49:2, Promega, Z529A). The supernatant was precipitated with twovolumes ethanol, and the pellet vacuum-dried and dissolved in 30 μL ofTE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA). The concentration of thedigested plasmid was then adjusted to 0.5 mg/mL by the addition of TEbuffer.

The kanamycin transcript was generated in the following reaction: 16 μLRNase-free water (Ambion, 9910G)  8 μL 10 × Transcription Buffer (8153G)40 μL  2 × Ribonucleotide Mix (8055G)  8 μL EcoR I cut plasmid (4 μg)  8μL 10 × Enzyme Mix (2079G) 80 μL

The reaction was incubated at 37° C. for one hour. Most of thesynthesized RNA contains a cap structure at the 5′ end (GpppG) because acap analogue was present in the Ribonucleotide Mix. Following completionof the reaction, 4 μL DNase I (Ambion, 2226G) were added and incubationcontinued for another 15 minutes at 37° C. One hundred-twentymicroliters of water and 100 μL of LiCl Precipitation Solution (Ambion,9480G) were then added. The reaction was chilled at −20° C. for 30minutes and centrifuged in a microcentrifuge at 14,000 rpm for 15minutes. The pellet was washed once in 70% ethanol and dissolved in 50μL of water. The concentration of the RNA was determinedspectrophotometrically assuming that a 1 mg/mL solution would provide anabsorbance of 25 at 260 nm.

The RNA was first prepared by attaching a small RNA oligonucleotide atthe 5′ end that served as a PCR anchor. In this way the entire 5′ end ofthe RNA could be amplified by the PCR. Prior to the ligation of this RNAoligo to the kanamycin RNA, the kanamycin RNA was first treated withcalf intestinal alkaline phosphatase (CIAP) and tobacco acidpyrophosphatase (TAP). The phosphattase step makes unavailable for theligation pathway any RNA molecules that do not contain a 5′ cap. Oncethe phosphatase is removed, the cap itself is removed with TAP. Thesynthetic kanamycin RNA was treated with CIAP in the following reaction: 0.6 μL 850 μg/mL total Mouse Liver RNA (Promega F160A)   1 μL 5 pg/μLcapped Kanamycin RNA   5 μL 10 × CIAP buffer   1 μL 40 U/μL rRNasin ®(Promega N251E)   2 μL 1 U/μL CIAP 40.4 μL water   50 μL

Following a one-hour 37° C. incubation, 250 μL water, 75 μL 10 Mammonium acetate and 375 μL phenol:chloroform:isoamyl alcohol (49:49:2)were added. The reaction was vortexed and phases separated by a 5 minutecentrifugation in a microcentrifuge. The supernatant (350 μL) wasremoved and the extraction repeated. The supernatant was precipitated bythe addition of 900 μL ethanol and centrifuged 5 minutes. Following a70% ethanol wash, the pellet was dissolved in 43.5 μL water. To theCIAP-treated RNA were added:

5 μL 10× TAP buffer (Epicentre)

1 μL 40 U/μL rRNasin®

1 μL 0.1 U/μL TAP (Epicentre, T19500)

Following a one-hour incubation at 37° C., the reaction was extractedand precipitated as above and the pellet dissolved in 13 μL of water. Tothat solution were then added:

4 μL 10× RNA Ligase buffer

1 μL 0.25 μg/μL RNA oligo 30-mer

1 μL 40 U/μL rRNasin®

1 μL 10 U/μL RNA Ligase (Promega, M1051)

20 μL 40% polyethylene glycol (SigmaP-2139)

and the reaction was incubated overnight (about 18 hours) at 16° C.

-   10× CIAP buffer: 100 mM Tris-HCl pH 8, 100 MM MgCl₂, 0.5 M NaCl, 10    mM DTT-   10× TAP buffer: 0.5M sodium acetate pH 6, 10 mM EDTA, 1%    beta-mercaptoethanol, 0.1% Triton X-100-   10× RNA Ligase buffer: 0.5 M Tris-HCl pH 8, 100 mM MgCl₂, 0.68%    beta-mercaptoethanol, 10 mM ATP

Following the ligation step, 250 μL of water, 75 μL of 10 M ammoniumacetate and 900 μL ethanol were added to the reaction. The mixture wasvortexed and then centrifuged 20 minutes in a microcentrifuge at 14,000rpm at 4° C. The pellet was washed in 70% ethanol and dissolved in 15 μLwater. CDNA was first synthesized from the RNA prior to PCR. To the RNAwas added 1 μL (50 pmoles) of a CDNA synthesis probe (SEQ ID NO:81) andthe mixture heated at 70° C. for 5 minutes, then cooled to roomtemperature for 10 minutes. To the RNA/probe mix were added:

-   -   5 μL 5× First Strand buffer (Promega, C121A)    -   1 μL 40 U/μL rRNasin®    -   2.5 μL 40 mM sodium pyrophosphate (Promega, C113A)    -   1 μL 25 U/μL AMV reverse transcriptase (Promega, M5108)

The reaction was incubated for 1 hour at 42° C. and then terminated bythe addition of 0.5 μL of 0.5 M EDTA and 74 μL water. To 5 μL of thecDNA (SEQ ID NO:77) were added:

-   -   5 μL 10× Thermophilic buffer (Promega, M190G)    -   5 μL 10× PCR dNTP    -   5 μL 25 mM MgCl₂ (Promega, A351H)    -   1 μL 320 μg/mL upstream probe(SEQ ID NO:78)    -   26.5 μL water

The reaction was mixed and covered with 50 μL mineral oil. The mixturewas put into a thermalcycler (Perkin-Elmer Model 480) at 95° C. After 2minutes, 1 μL of 5 U/μL Tag DNA polymerase (Promega, M166B) was addedand the reaction cycled 95° C. for 1 minute, 43° C. for 1 minute, 72° C.for 2 minutes for 5 cycles and then brought to 85° C. Then, 1 μL of 320μg/ml of downstream probe (SEQ ID NO:79) was added and the reactioncycled 95° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 1 minutefor 30 cycles followed by 5 minutes at 72° C. then 4° C. 10× PCR dNTP: 1mM each of dATP, dGTP, dCTP and dTTP The PCR reaction generated a 613 bpproduct. To remove unincorporated probes and dNTP, a 15 μl aliquot ofthe PCR reaction was purified with Promega's Wizard™ PCR Preps (A7170)according to kit instructions. The concentration of the purified PCRproduct was determined by a pyrophosphorolysis assay. A Master Mix (MM)was assembled containing the following components:  20 μL 10 × Buffer A(Promega, R001A)  2 μL 40 mM sodium pyrophosphate  2 μL 10 μM ADP (SigmaA5285)  5 μL 1 U/μL NDPK (Sigma N0379) 151 μL water 180 μL

This mix was used in reactions containing control PhiX 174 HinF I DNAstandard (Promega G175A) or aliquots of the PCR reaction, with andwithout added 4 DNAP (10U). The PCR reaction was diluted 10× TE buffer(10 mM Tris, 1 mM EDTA) for use in the assay. The results are shownbelow. PhiX 1 ng/μL DNA PCR T4 DNAP LU 1 18 μL — — + 0.950 2 18 μL 1 μL— + 44.92 3 18 μL 2 μL — + 68.86 4 18 μL 3 μL — + 90.88 5 18 μL — 1 μL −1.244 6 18 μL — 2 μL − 1.388 7 18 μL — 1 μL + 47.97 8 18 μL — 2 μL +68.69

Light units (LU) generated resulted from adding 2 μl of reaction mixesto 100 μl of L/L reagent and measuring on a Turner® TD20/20 luminometer.

As can be seen, the PhiX 174 HinF I DNA produced a light signal that wasproportional to the amount of DNA added. The concentration of theten-fold diluted PCR reaction is almost exactly that of the DNAstandard, so the undiluted PCR product DNA is at a concentration of 10ng/μL. Note that only background light units are seen for the reactionsthat contained the PCR product but no T4 DNAP. This indicatesessentially complete removal of the dNTP's during clean-up on theWizard™ resin.

Next, the PCR product was detected by hybridizing andpyrophosphorolyzing a probe that bound to internal sequences. Thesequence of the probe was 5′ GCAACGCTACCTTTGCCATGTTTC 3′. (SEQ ID NO:80)

For this purpose it was found most suitable to use the Klenow exo−polymerase (Promega, M218B) in place of T4 DNAP. A 2× Master Mix (MM)was assembled as below:  60 μL 10 × DNAP buffer (Promega, M195A)  15 μL40 mM Sodium Pyrophosphate  15 μL 10 U/μL Klenow exo− polymerase  3 μL 1U/μL NDPK  6 μL 10 μM ADP 201 μL water 300 μL

PCR product (between 0 and 20 ng) was mixed (or not) with 1 μL of 1μg/μL probe and water as shown below. The mixtures were heated at 95° C.for 3 minutes and then permitted to cool to room temperature for 10minutes. Then 20 μL of 2× Master Mix were added and the reactionsincubated for 20 minutes at 37° C. before adding 4 μL to 100 μL of L/Lreagent (Promega, F202A). PCR Target Probe Water LU 1   20 ng − 18 μL37.84 2   20 ng + 17 μL 423.2 3   10 ng − 19 μL 16.51 4   10 ng + 18 μL366.1 5   5 ng − 19 μL 7.79 6   5 ng + 18 μL 226.0 7  2.5 ng − 19 μL4.994 8  2.5 ng + 18 μL 171.2 9 1.25 ng − 19 μL 3.176 10 1.25 ng + 18 μL85.6 11   0 ng + 19 μL 2.656

It can be seen that substantially higher LU result when the probe ispresent along with the target DNA and that this signal is not due to theprobe alone (reaction 11). It is surprising that only a very smallamount of the PCR product target has re-annealed to give a signal duringthe course of the asssay, even at the higher DNA amounts. The lightunits generated represent only one tenth of the DNA that was added tothe reactions and from the data it is apparent that about 10 pg of thePCR product can easily be detected in a probe-dependent manner.Kanamycin RNA Oligo 5′ AGAGUCUUGACGGAUCCAGGUACCAGUAAA 3′ SEQ ID NO:77Upstream probe: 5′ TGATCGTAAGAGTCTTGACGGATC 3′ SEQ ID NO:78 Downstreamprobe: 5′ TCATTCGTGATTGCGCCTGAGCGA 3′ SEQ ID NO:79 Internal Probe: 5′GCAACGCTACCTTTGCCATGTTTC 3′ SEQ ID NO:80 cDNA Synthesis Primer: 5′AAATCACTCGCATCAACCAAACCG 3′ SEQ ID NO:81

EXAMPLE 22 Mismatch (Mutation) Detection in a Target PCR Product

To demonstrate base interrogation, or mismatch detection, four differentprobes were used to hybridize with the above PCR product. The wild-type(WT) probe was the same as that used in Example 9, or Probe 1 (SEQ IDNO:48). In addition, three additional probes were used that differed intheir terminal base at the 3′ end.

The WT probe (Probe 1) contained a C that matched to the G present onthe PCR template. Three additional probes contained either G, A or T atthe 3′-terminal position (Probe 2, (SEQ ID NO:49), Probe 3 (SEQ IDNO:50) and Probe 4 (SEQ ID NO:51) respectively) and thus when hybridizedto the template created mismatches of GG, GA and GT, respectively. Thesemismatched bases substantially block pyrophosphorolysis of thehybridized probe:target, permitting determination of which base ispresent on the DNA template at that position.

A 2× Master Mix (2×MM) was prepared as in Example 21 and 1 μL of 10ng/μL target PCR product were mixed with 1 μL of 1 μg/μL probe and wateror TE buffer (10 mM Tris, 1 mM EDTA) as below. The mixtures were heatedfor 3 minutes at 95° C. then permitted to cool to room temperature for10 minutes. Then, 20 μL of 2×MM were added, the reactions mixed andpermitted to incubate for 20 minutes at 37° C. to hybridize and formtreated samples prior to adding 4 μL of each to 100 μL of L/L reagent(Promega, F202A) and measuring the resulting light units produced.Target PCR/ Water TE Probe # (μL) (μL) LU 1 +/− 18 1 21.82 2 −/1 19 —2.628 3 +/1 18 — 322.1 4 +/− 18 1 14.69 5 −/2 19 — 3.277 6 +/2 18 —57.44 7 +/− 18 1 23.14 8 −/3 19 — 4.861 9 +/3 18 — 40.90 10 +/− 18 114.98 11 −/4 19 — 5.899 12 +/4 18 — 43.33

It can be seen that the greatest LU were obtained in the case of thematched probe (Probe 1). Subtracting the backgrounds of PCR productalone and probe alone, the following LU were obtained in the case of thematched and mismatched probes: Probe And Template Resulting In LU GCmatch 297.7 GG mismatch 39.47 GA mismatch 12.90 GT mismatch 22.45

It is clear that having a probe 3′-terminal base that mismatches withthe target dramatically reduces the rate of pyrophosphorolysis of thehybridized probe.

The probe alone backgrounds seen above are low (<10 LU). However, probeshave been encountered that give very high backgrounds (as much as 500 LUper 100 ng probe). Such probes are generally complementary and capableof forming either self-dimers or hairpin structures leading todouble-stranded regions at their 3′ ends. Such probes are to be avoidedand can often be detected using various secondary structure predictionprograms. If one set of probes provides high background, it may bepossible to use adjacent probes to the other strand with their 3′ endsinterrogating the same site. Probe 1 SEQ ID 5′ GCAACGCTACCTTTGCCATGTTTC3′ NO:48 Probe 2 SEQ ID 5′ GCAACGCTACCTTTGCCATGTTTG 3′ NO:49 Probe 3 SEQID 5′ GCAACGCTACCTTTGCCATGTTTA 3′ NO:50 Probe 4 SEQ ID 5′GCAACGCTACCTTTGCCATGTTTT 3′ NO:51

EXAMPLE 23 Mutation Detection on Pseudo-PCR Product Synthetic Targets

In order to show base interrogation on DNA targets where a base hasactually been changed (mutation), synthetic oligonucleotides were madethat correspond to a region of cytomegalovirus DNA in which a mutationcan be present that has been shown to be responsible for resistance tothe drug gancyclovir.

The upper strand of the wild type target corresponds to sequence 1 below(SEQ ID NO:82) the bottom strand corresponds to sequence 2 below (SEQ IDNO:83). The base that is mutated is indicated in bold type. The upperstrand of the mutant target (sequence 3 below, (SEQ ID NO:84)) is thesame as sequence 1 but the bolded base has been changed from an A to aG. The bottom strand of the mutant target (sequence 4 below, (SEQ IDNO:85)) is the same as sequence 2 but the bolded base has been changedfrom a T to a C.

Two oligonucleotide probes were used to interrogate the position of themutated base, one corresponding to the wild type and the other to themutant sequence. The sequence of the wild type interrogation probe issequence 5 (SEQ ID NO:86) below and the sequence of the mutantinterrogation probe is sequence 6 below (SEQ ID NO:35). These probeswere identified by the numbers 9211 and 9212 respectively. Sequence 5differs from sequence 6 at the position of the bolded base and for theseprobes, the mismatched base is three nucleotides in from the 3′ end ofeach probe.

It was expected that the wild type probe would give the strongest signalon the wild type target and the mutant probe the strongest signal on themutant target. This was found to be the case as demonstrated in thestudy detailed below.

Wild type and mutant DNA targets to be interrogated were assembled bymixing together sequence 1 with sequence 2 and sequence 3 with sequence4 to a final concentration of 0.3 μg/mL. Interrogation probes 9211 and9212 were both dissolved to a concentration of 1 mg/mL in TE buffer (10mM Tris, 1 mM EDTA). Reactions were assembled as below and containedeither target only, probe only or target plus probe: Target 9211 9212Water Wild Type (μL) (μL) (μL) (μL) 1 and 2 1 — — 19 3 and 4 — 1 — 19 5and 6 — — 1 19 7 and 8 1 1 — 18 9 and 10 1 — 1 18

Reactions were mixed and heated at 95° C. for 3 minutes and thenpermitted to cool to room temperature for 10 minutes on the bench tohybridize and form treated samples. Then 20 μL of a 2× Master Mix wereadded, the reactions incubated 20 minutes at 37° C. then 4 μL of eachwere added to 100 μL of L/L reagent the resulting light units determinedin a luminometer.

Master Mix:

-   -   60 μL 10× DNAP buffer    -   15 μL 40 mM sodium pyrophosphate    -   15 μL Klenow exo− DNAP    -   3 μL 1 U/μL NDPK    -   6 μL 10 μM ADP    -   201 μL water

The resulting relative light units were as follows. Reaction Light Units1 1.687 2 1.732 3 4.313 4 3.948 5 10.54 6 10.04 7 220.8 8 206.8 9 49.6710 37.33It can be seen (1 and 2) that the DNA target itself yields very few LUas is the case for the interrogation probes alone (3 through 6). Wildtype interrogation probe mixed with wild type target provided over 200LU, whereas the mutant probe mixed with the wild type target providedless than 50 LU. After substituting the backgrounds given by the targetand probes alone, it can be seen that the wild type pronbe providedroughly five-fold more signal on the wild type target than did themutant probe.

The above study was then repeated but substituting the mutant DNA targetfor the wild type target. The resulting LU are shown below. ReactionLight Units 1 1.760 2 1.779 3 4.157 4 4.316 5 11.0 6 10.56 7 34.31 829.53 9 241.9 10 264.5

Again, it can be seen that background light units provided by targetalone and probes alone are low (1-6) and that this time the greatestsignal was seen with the mutant (9212) probe instead of the wild typeprobe. Thus, by comparing the ratio of signals obtained with the wildtype and mutant probes, one can distinguish the wild type from themutant DNA. PCR SEQ ID 5′ CGTGTATGCCACTTTGATATTACACCCATGAAC SequenceNO:82 GTGCTCATCGACGTGAACCCGCACAACGAGCT 3′ 1 PCR SEQ ID 5′CGTTGTGCGGGTTCACGTCGATGAGCACGTTCA Sequence NO:83TGGGTGTAATATCAAAGTGGCATACACGAGCT 3′ 2 PCR SEQ ID 5′CGTGTATGCCACTTTGATATTACACCCGTGAAC Sequence NO:84GTGCTCATCGACGTGAACCCGCACAACGAGCT 3′ 3 PCR SEQ ID 5′CGTTGTGCGGGTTCACGTCGATGAGCACGTTCA Sequence NO:85CGGGTGTAATATCAAAGTGGCATACACGAGCT 3′ 4 PCR SEQ ID 5′CACTTTGATATTACACCCATG 3′ Sequence NO:86 5 (9211) PCR SEQ ID 5′CACTTTGATATTACACCCGTG 3′ Sequence NO:35 6 (9212)

EXAMPLE 24 Cloning and Expression of a Gene Encoding a NDPK Enzyme fromThermophilic Bacteria Pyrococcus furiosis

The cloning and expression of a gene from the thermophilic bacteriaPyrococcus furiosis [Pfu; Pfu-Vcl(DSM 3638)] is described in thisExample. This gene encodes the nucleoside diphosphate kinase (NDPK)enzyme. The protein originates from a thermophile and remains active atelevated temperatures for a longer period of time than the correspondingprotein from a mesophilic organism. The protein also remains active atroom temperature longer than the corresponding mesophilic enzyme. If theprotein were stable at elevated temperature, it could function incombination with a thermostable polymerase in a pyrophosphorylationreaction, thereby eliminating the need to carry out separatepyrophosphorylation and phosphate transfer steps as needed for the NDPKderived from yeast.

The amino acid sequences of known NDPKs (Gene 129:141-146, 1993 and theNCBI sequence of NDPK from Pyrococcus horikoshii) were compared andsegments of high amino acid homology identified.

Two degenerate DNA primers were designed that would permit the DNAbetween them to be amplified. These primers, Pf1 (SEQ ID NO:87) and Pf2(SEQ ID NO:87), are shown below, and were dissolved in TE buffer (10 mMTris, 1 mM EDTA). A ‘6’ in the primer sequence indicates an inosine thatcan hybridize to any base.

Chromosomal DNA from Pfu was isolated by resuspending frozen cell pastefrom a 3 mL overnight (about 18 hours) culture pellet in 1 mL TE buffer(10 mM Tris, 1 mM EDTA), lysing the cells by beating with zircon beads,followed by two phenol extractions and a chloroform extraction. The DNAin the supernatant was then ethanol precipitated, dried, and resuspendedin TE buffer overnight (about 18 hours). The resuspended DNA was treatedwith 20 units of RNaseI, reprecipitated and resuspended in TE buffer.

The Pfu genomic DNA was used in the following DNA amplificationreaction.  2 μL 1.5 μg Pfu DNA (pre-denatured for 5 minutes at 99° C.,then placed on ice)  5 μL PCR buffer  4 μL 25 mM MgCl₂  2 μL each primerPf1 and Pf2 (200 picomoles each)  1 μL 10 mM dNTP mix 25 μL water  1 μLTaq (5 units) 10 μL 5M betaine

Different extension temperatures in the range from 41° C. to 55° C. weretested in the following PCR profile: 94° C., 2 minutes; (94° C., 15 or40 seconds; 45° C. to 55° C., 45 or 90 seconds; 72° C., 1 or 2minutes)×20; 72° C. 2 minutes. The profile varied for the differentextension temperatures, with 41° C. and 43° C. extension temperatureshaving the lesser times, and the remaining extension temperatures havingthe longer times.

The reaction products were analyzed by gel electrophoresis on a 1.2% TBEagarose gel. The products of the reaction were detected by staining thegel with ethidium bromide and photographing the gel under UV light. A300 bp DNA fragment was identified as the product of the reaction andwas present to a greater extent when using extension temperatures from41° C. to 47° C. The 300 bp fragment was gel purified (Promega, A7170)and cloned into PGEM-T vector (Promega, A3600).

The sequence of the insert was determined and found to encode an openreading frame. The translated amino acid sequence of this open readingframe matched the protein sequence of the Pyrococcus horikoshii NDPKgene with 94% homology.

A hybridization probe, Pf3 (SEQ ID NO:89), was designed from thesequence obtained. This probe was ³²P labeled and used to identify thesize of the DNA fragments encoding the corresponding gene in chromosomaldigests of the DNA from Pfu using standard Southern blot hybridizationmethods.

A size-specific EcoR I library of DNA fragments from Pfu was produced bydigesting Pfu chromosomal DNA with EcoR I, fractionating the DNAfragments using agarose gel electrophoresis, identifying the segment ofthe fractionated DNA that corresponded to the 2 Kb EcoR I fragmentidentified as containing the desired gene and isolating the DNA from thegel. The DNA isolated was cloned into plasmid pZERO2 (Invitrogen), andthe resulting library was transformed into E. coli TOP10 (Invitrogen).The transformants were probed using the same probe employed duringSouthern hybridization and two clones were identified as potentialcandidate clones. From this analysis, an EcoR I fragment about 2 kb insize was identified as a target for additional cloning.

The sequences of the two candidate clones were found to contain theexact sequence present in the 300 bp DNA segments sequenced earlier inaddition to DNA sequences both 5′ and 3′ to that sequence. The openreading frame identified earlier was found to extend significantlybeyond the limits of the 300 bp segment sequenced earlier. Theadditional segments of the open reading frame again showed good homologywith NDPK. The Pfu NDPK nucleotide sequence is identified as SequencePf4 (SEQ ID NO:90) and the corresponding amino acid sequence isidentified as Pf5 (SEQ ID NO:91). The protein codes for 161 amino acidresidues.

The coding segments of the gene were amplified using primers Pf6 (SEQ IDNO:92) and Pf7 (SEQ ID NO:93), and placed into a high protein expressionvector for E. coli JHEX25, an IPTG inducible promoter system (Promega).Bacterial transformants were grown in LB media and induced for proteinexpression. Samples of the induced bacterial cultures were boiled in 2×SDS Sample buffer and loaded onto an SDS gel. After running, the gel wasstained with Coomassie Blue.

After destaining in 1% acetic acid and 10% methanol, the lanescontaining extracts from cells with the open reading frame were found tocontain a large amount of a protein of about 14 Kd, the expected size ofthe gene product from the insert.

Then, a comparison of the open reading frame to the published sequenceof the Pfu genome (University of Utah) was performed and the openreading frame was found to exactly match a region of the genome of thisorganism as expected. Pf1 5′ AT6AT6AA(AG)CC6GA(TC)G(GC)6GT 3′ SEQ IDNO:87 Pf2 5′ AA(AG)TC6CC6C(TG)6AT6GT6CC6GG 3′ SEQ ID NO:88 Pf3 5′GAGAAGCACTATGAGGAGCAC 3′ SEQ ID NO:89 Pf4 5′ATGAACGAAGTTGAAAGAACATTGGTAATCATAAAGCCCGACGCAGTAGTT SEQ ID NO:90AGGGGTCTAATAGGTGAAATTATAAGCAGGTTTGAGAAGAAAGGCCTCAAGATTGTTGGAATGAAGATGATCTGGATAGACAGGGAGTTGGCTGAGAAGCACTATGAGGAGCACAAAGGAAAGCCCTTCTTTGAGGCTCTCATAGATTACATAACGAAAGCTCCAGTAGTTGTTATGGTGGTTGAGGGAAGGTATGCAGTAGAAGTAGTTAGAAAGATGGCTGGAGCTACTGATCCAAAGGACGCAGCACCTGGGACAATTAGGGGAGATTATGGACTTGACATAGGAGATGCAATCTACAACGTGATTCATGCCAGTGATTCAAAGGAAAGTGCGGAGAGGGAAATAAGCCTGTACTTTAAACCTGAAGAAATTTATGAATACTGCAAAGCTGCAGATTGGTTTTACAGGGAAAAGAAG CAGGCTAAATGCTGA 3′Pf5 MNEVERTLVIIKPDAVVRGLIGEIISRFEKKGLKIVGMKMIWIDRELAEKHYE SEQ ID NO:91EHKGKPFFEALIDYITKAPVVVMVVEGRYAVEVVRKMAGATDPKDAAPGTIRGDYGLDIGDAIYNVIHASDSKESAEREISLYFKPEEIYEYCKAADWFYREKKQA KC Pf6 5′GGGTGCTTTTCATGAACGAAGTTGA 3′ SEQ ID NO:92 Pf7 5′AAGGGCAAAAATTCTAGAGTTCAGCAT 3′ SEQ ID NO:93

EXAMPLE 25 Purification of Cloned Pfu NDPK Protein from E. coli

An initial fermentation of Top10 E. coli cells expressing the Pfu NDPKprotein, as described in Example 24 yielded about 10 g of wet cellpaste. The protein purification scheme was essentially that as describedin Kim, S. et al. Molecules and Cells, 7:630, 1997. One gram of cellpaste was resuspended in 10 mL of 20 mM Tris-acetate pH 7.4/1 mM EDTA/2μg/mL aprotinin/0.1 mg/mL lysozyme and incubated at room temperature for10 minutes. The suspension was then sonicated for 2 minutes at 50%cycle, held on ice for 5 minutes, then sonicated an additional 2minutes. The suspension was centrifuged at 15,000×g for 20 minutes at 4°C. and the supernatant transferred to a new tube.

The supernatant was heated to 80° C. for 20 minutes to denaturenon-thermostable proteins. Precipitant was pelleted by centrifugation at14,000×g for 20 minutes at 4° C. and supernatant was transferred to anew tube.

Ten milliliters of supernatant were applied to a 5 mL ATP-sepharose(Sigma, A-9264) affinity column equilibrated with Buffer A (20 mMTris-acetate pH 7.4/20 mM NaCl/0.1 mM EDTA/3 mM MgCl₂/15 mM BME). Theflow through was collected by gravity. The column was washed with 6column volumes of Buffer B (Buffer A containing 500 mM NaCl). The boundprotein was eluted in two steps: 5 mL Buffer B+1 mM dCTP (Promega,U122A) followed by 5 mL of Buffer B+1 mM ATP (Sigma, A-7699).

SDS-PAGE analysis of the purification fractions showed a large loss oftotal protein following the heat denaturation step, with the NDPK beingthe major band loaded on the column. About 50% of the loaded NDPK was inthe flow-through fraction. Eluted NDPK appeared in both the dCTP and ATPelutions at greater than 80% purity.

EXAMPLE 26 Thermostable NDPK Activity Assays Activity Assay

The activity assay for NDPK measures ATP created following phosphatetransfer from dCTP to ADP. A linear range for the amount of enzyme wasdetermined using yeast NDPK in a 10 minute assay at 37° C. and was foundto be 0.002-0.0002 units, or 0.012-1.2 ng protein. The optimalconcentrations of ADP and dCTP in the assay were found to be 100 nM and10 μM respectively, in order to give Turner® TD20/20 luminometerreadings within a readable scale.

The Pfu NDPK activity in the dCTP and ATP eluted fractions, as describedin Example 25, was examined after extensive dialysis of the fractions toremove nucleotides. Both the dCTP and ATP elutions of the purified PfuNDPK were also passed over a De-Salt™ column (Pierce, 43230) accordingto manufacturer's recommendations to further remove excess nucleotides.

Activity of Pfu NDPK was measured in a 10 minute assay at both 37° C.and 70° C. Activity was observed at both temperatures. If full enzymaticactivity is presumed at the 70° C. optimum, then about 40% of thatactivity was seen at the lower temperature. The estimated unit activityfor the fractions was determined by comparison of the light output,resulting from ATP formation, of yeast NDPK at 37° C. with the lightoutput of the Pfu NDPK at 70° C. For example: if 0.0002 units of yeastNDPK provides 7000 relative light units after 10 minutes at 37° C., then0.0002 units of Pfu NDPK is presumed to provide 7000 relative lightunits after 10 minutes at 70° C.

Using this unit equivalency based on light output, the dCTP and ATP PfuNDPK fractions were assigned a unit activity of 0.5 units/μL.

Activity Assay at Two Temperatures

The activity levels of the Pfu NDPK from both the ATP- and thedCTP-eluted fractions were compared to the activity level of the yeastNDPK at both 70° C. and 37° C. A series of 10-fold serial dilutions ofthe three enzyme solutions was made in Nanopure water to a finaldilution of 1:10,000. The following master mix was prepared: 889 μLNanopure water (Promega AA399) 100 μL 10 × DNAP Buffer (Promega M195A) 10 μL 10 μM ADP (Promega, A-5285)  1 μL 10 μM dCTP (Promega, U128B)

Into each reaction tube were placed 180 μL master mix preheated toeither 37° C. or 70° C. and 20 μL of each 1:10,000 NDPK dilution, andtubes were incubated at the indicated temperature. Twenty microlitersamples were removed at various time points, added to 100 μL of L/Lreagent (Promega, F202A) and light units read in a TMDE™ luminometer.The t=0 time point was never incubated at elevated temperatures and wasplaced on ice. The following data were obtained: Time (minutes) 70° C.37° C. Yeast 0 4896 NDPK 1.0 5126 2.5 5163 5 6946 10 6687 7503 15 673520 6806 25 7298 Pfu 0 327 NDPK 1.0 749 (dCTP) 2.5 1772 5 2794 10 3191111 15 4364 20 5025 25 5830 Pfu 0 1255 NDPK 1.0 2235 (ATP) 2.5 4410 55925 10 6039 973 15 6828 20 7747 25 10026 No NDPK 0 56

The Pfu NDPK is more active at 70° C. than at 37° C. This is evident bycomparing relative light units at 10 minutes activity at 37° C. and 70°C. (1:10,000 dilution). There is about 10-fold more yeast protein in the70° C. reaction than Pfu protein as determined by a standard Bradfordassay. Therefore, the Pfu NDPK enzyme may have a higher specificactivity. For the 37° C. assay, the yeast NDPK was further diluted to1:100,000 and produced 247 light units at this dilution. The light unitsincreased slightly and then leveled off for the yeast enzyme, suggestingthermal inactivation of the enzyme at 70° C. The Pfu NDPK light outputincreased over time.

Thermostability of Pfu NDPK and Yeast NDPK

Yeast NDPK stock at 1 unit/μL was serially diluted in Nanopure water toa 1:100,000 final dilution. The dCTP- and ATP-eluted Pfu NDPK stock wereserially diluted in Nanopure water to a 1:10,000 final dilution. Thisequalized the amount of protein present in the Pfu NDPK and Yeast NDPKfinal dilutions, as determined by standard Bradford protein assay andSDS-PAGE analysis.

Two microliters of the diluted enzymes were added to 18 μL of master mixin chilled tubes and placed on ice. These are the t=0 time points. Theremainder of the diluted NDPK solutions were pre-warmed at 70° C. Foreach time point, a 2 μL aliquot of the enzyme dilution was added to 18μL master mix and then placed on ice. After the t=10 minutes time point,all tubes were incubated at 37° C. for 10 minutes. Then, 100 μL of L/Lreagent were added to each reaction and the relative light unitsmeasured on a TMDE™ luminometer. The following results were obtained.Relative Light Units Time Pfu NDPK Pfu NDPK (minutes) Yeast (dCTP) (ATP)0 1877 446 615 1 263 358 400 2 47 299 472 3 42 319 446 4 40 296 432 5 38315 353 7.5 36 241 339 10 37 239 307

The yeast NDPK appears to be thermolabile, whereas the Pfu NDPK isrelatively thermostable. The purified Pfu NDPK had a half-life of about10 minutes, whereas the yeast NDPK had a half-life of about 0.6 minutes.

EXAMPLE 27 Interrogation Assay at 70° C. Using Pfu NDPK and Tne TripleMutant DNA Polymerase

This Example uses thermostable NDPK and thermostable polymerase in aone-step 70° C. interrogation reaction. Synthetic CMV targets, differingat one nucleotide, were prepared by annealing single-stranded DNAs asdescribed in Example 2. The wild-type single-stranded DNAs used wereCV11 (SEQ ID NO:8) and CV12 (SEQ ID NO:9), and the mutantsingle-stranded DNAs used were CV 13 (SEQ ID NO:10) and CV 14 (SEQ IDNO:11). Interrogation oligonucleotides used were 9211 (SEQ ID NO:86) and9212 (SEQ ID NO:35).

The following solutions, brought to a final volume of 20 μL with water,were assembled and assayed: CMV CMV WT Mutant Interrogation Soln.*Target Target Oligo (1 μg) (0.1 U Tne) (0.2 U Tne) 1 — — — 48 72 2 5 ng— — 60 79 3 — 5 ng — 27 74 4 — — 9211 55 71 5 — — 9212 55 70 6 5 ng —9211 374* 599 7 5 ng — 9212  28* 80 8 — 5 ng 9211  53* 82 9 — 5 ng 9212180* 443*Soln. = solution; Average of two reactions.

The solutions were heated to 95° C. for 5 minutes, then cooled to roomtemperature for 10 minutes. Then 20 μL 2× master mix were added to eachreaction, and the reactions were incubated at 70° C. for 10 minutes. The2× master mix contains 100 μL 10× Thermophilic DNA Pol buffer (Promega,M190A), 100 μL 25 mM MgCl₂ (Promega, A351B), 25 μL 40 mM NaPPi (Promega,E350B), 10 μL 10 μM ADP (Promega, A5285), 25 units Tne triple mutant(Promega), 2.5 units Pfu NDPK, and 259.4 μL Nanopure water. Eachreaction contained 1 unit Tne DNA Polymerase and 0.1 units Pfu NDPK. Anidentical set of reactions was also performed containing 0.2 units PfuNDPK per reaction. Four microliters of each reaction were then added to100 μL of L/L reagent and relative light units (rlu) were measured on aTMDE™ luminometer.

The results obtained indicate that Pfu NDPK is active in thisinterrogation assay at 70° C. Doubling the NDPK concentration increasedthe light output to levels seen previously with 0.1 units yeast NDPK at37° C. CV11 5′ CGCTTCTACCACGAATGCTCGCAGACCATGCTGCACGAATACGTCAGAAAG SEQID NO:8 AACGTGGAGCGTCTGTTCGAGCT 3′ CV12 5′CCAACAGACGCTCCACGTTCTTTCTGACGTATTCGTGCAGCATGGTCTGCG SEQ TD NO:9AGCATTCGTGGTAGAAGCGAGCT 3′ CV13 5′CGCTTCTACCACGAATGCTCGCAGATCATGCTGCACGAATACGTCAGAAA SEQ ID NO:10GAACGTGGAGCGTCTGTTCGAGCT 3′ CV14 5′CCAACAGACGCTCCACGTTCTTTCTGACGTATTCGTGCAGCATGATCTGCG SEQ ID NO:11AGCATTCGTGGTAGAAGCGAGCT 3′ 9211 5′ CACTTTGATATTACACCCATG 3′ SEQ ID NO:869212 5′ CACTTTGATATTACACCCGTG 3′ SEQ ID NO:35

EXAMPLE 28 One Step Interrogation on β-globin PCR Targets: Comparison of70° C. Reaction Using Tne Triple Mutant Polymerase and Pfu NDPK to 37°C. Reaction Using Klenow Exo− Polymerase and Yeast NDPK

A method was carried out using a thermostable polymerase (Tne triplemutant) and thermostable NDPK (Pfu) and compared to more thermallylabile polymerase (Klenow exo−) and NDPK (yeast) to assay for native andmutant sequences of β-globin. These methods were carried out at 37° C.and 70° C. As will be seen from the results that follow, use of thethermostable enzymes permit a one step (or one-pot) reaction in whichdepolymerization and ATP formation are carried out at an elevatedtemperature. The DNA interrogation probes used were 9994 (SEQ ID NO:94),9995 (SEQ ID NO:95), 10665 (SEQ ID NO:96), and 11472 (SEQ ID NO:97).Probes 9994 and 9995 interrogate the TCTT site. Probes 10665 and 11472interrogate the 17 (A to T) site. PCR probes used were Probe 9992 (SEQID NO:98) and 9993 (SEQ ID NO:99). Reaction conditions are as describedin Example 31.

Two sets of the following reactions were assembled and brought to afinal 20 μL volume with nanopure water: Interrogation Oligo rlu rluReaction Target 1 μl/1 μg (70° C.) (37° C.) 1 — —  41  5 2 WT* — 165  943 — WT 9994  51  12 4 — Mut 9995*  42  6 5 — WT 10665  45  14 6 — Mut11472  44  6 7 WT WT 9994 645* 360* 8 WT Mut 9995 199* 109* 9 WT WT10665 525* 233* 10 WT Mut 11472 169* 121* WT = wild type; Mut == mutant;Average of two values. 2 × Master 2 × Master Mix for 37° C. Mix for 70°C. 10 × DNAP 60 μL(Promega, 100 μL(Promega buffer M195A) M190A) Klenowexo-  3.75 units — Tne triple —   25 units mutant 40 mM NaPPi   7.5 μL  25 μL yeast NDPK    3 units — Pfu NDPK —  2.5 units 10 μM ADP   6.0 μL  10 μL Nanopure 219.75 μL 259.4 μL water

Four microliters of target solution were used for the set of 37° C.reactions, 8 μL target solutions were used for the set of 70° C.reactions. The assembled reactions were heated to 95° C. for 5 minutesand then cooled to room temperature for 10 minutes. Twenty microlitersof the 2× master mix were then added. The 37° C. reaction set wasincubated at 37° C. for 15 minutes, the 70° C. reaction set wasincubated at 70° C. for 5 minutes. Four microliters of each reactionwere added to 100 μL of L/L reagent (Promega, F202A) and light outputwas immediately measured on a TMDE™ luminometer.

The high temperature interrogation conditions improve the discriminationratios between wild type and mutant for the 17 (A to T) site primarilyby reducing the background signal from the mismatch. Discriminationratios at the TCTT site are essentially the same between the twotemperatures.

Interrogation Probes: 9994 5′ CCCTTGGACCCAGAGGTTCT 3′ SEQ ID NO:94 99955′ CCCTTGGACCCAGAGGTTGA 3′ SEQ ID NO:95 10665 5′ CTTCATCCACGTTCACCTTG 3′SEQ ID NO:96 11472 5′ CTTCATCCACGTTCACCTAG 3′ SEQ ID NO:97

PCR Target Probes: 9992 5′ GTACGGCTGTCATCACTTAGACCTCA 3′ SEQ ID NO:989993 5′ TGCAGCTTGTCACAAGTGCAGCTCACT 3′ SEQ ID NO:99

EXAMPLE 29 Detection of DNA Sequences in the Genome of Campylobacterjejuni

Oligonucleotides 11453 (SEQ ID NO:100) and 11454 (SEQ ID NO:101) areexactly complementary and can be annealed, thereby forming a synthetictarget representing a 70 bp segment of Campylobacter jejuni. These twooligonucleotides were diluted in nanopure water to a final concentrationof 10 μg/mL. Four microliters of each were then mixed with 232 μL 10 mMTris pH7.3 to yield a target solution of 0.3 μg/mL of DNA.Oligonucleotides 11451 (SEQ ID NO:102) and 11450 (SEQ ID NO:103) areCampylobacter jejuni interrogation probes that bind to opposite strandsof the bacterial genome represented in the synthetic target.oligonucleotide 11451 anneals to oligonucleotide 11454. Oligonucleotide11450 anneals to oligonucleotide 11453.

The following solutions were assembled in triplicate and nanopure wateradded to a final volume of 20 μL. 0.3 ng 1 μg Solution Target Proberlu 1. + 11451 391 2. + 11450 241 3. + none 28 4. − 11451 248 5. − 1145030 6. − none 24

The assembled solutions were incubated at 92° C. for 5 minutes, thencooled at room temperature for 10 minutes. Master mix was prepared as inExample 1 using 10 units Klenow exo− polymerase and 4 units NDPK. Twentymicroliters of master mix were added to each tube and incubated at 37°C. for 15 minutes. Five microliters of each solution were then combinedwith 100 μL of L/L reagent (Promega F202A) and light output measuredimmediately on a Turner® TD20/20 luminometer. The average relative lightunits (rlu) are recorded in the table above

Using each of the interrogation probes with the target appears to givestrong net signal. The top probe (11451) however, gives very strongsignal alone, possibly due to hairpin formation, and is less suitablefor interrogation. The bottom interrogation probe (11450) is the betterfor interrogation. 11453 5′CTTGAAGCATAGTTCTTGTTTTTAAACTTTGTCCATCTTGAGCCGCTTGA SEQ ID NO: 100GTTGCCTTAGTTTTAATAGT 3′ 11454 5′ACTATTAAACTAAGGCAACTCAAGCGGCTCAAGATGGACAAAGTTTA SEQ ID NO: 101AAAACAAGAACTATGCTTCAAG 3′ 11451 5′ AGTTCTTGTTTTTAAACTTTGTCCATCTTG 3′ SEQID NO: 102 11450 5′ CAAGATGGACAAAGTTTAAAAACAAGAACT 3′ SEQ ID NO: 103

EXAMPLE 30 Enhancing Output Discrimination by DestabilizingInterrogation Probes with Internal Mismatches

Prothrombin PCR fragments were interrogated to determine if theycontained a single nucleotide polymorphism (SNP) associated withprothrombin as described in Example 35. The interrogation probes weredesigned to compare data when there is a potential for a mismatchednucleotide only at the 3′-terminal base of the interrogation probeversus an interrogation probe having this same potential mismatch and anadditional mismatch 9 bases from the 3′ end.

Probes PT5 and PT6 (SEQ ID NO:104 and SEQ ID NO:105, respectively) wereused to PCR amplify a region of human genomic DNA spanning about 500base pairs encoding the prothrombin gene. The PT5 probe hasphosphothioate linkages between the first five bases at the 5′ end. Asdescribed in Example 38, below, these linkages, present on one strand ofthe resulting PCR product, are resistant to cleavage with T7 PolymeraseExonuclease 6 (Exo 6). The PCR reaction was set up as follows:  10 μL 10× PCR buffer   6 μL 25 mM MgCl2   2 μL 10 mM dNTP mixture (2.5 mM eachdNTP)   2 μL 100 pmoles probe PT5   2 μL 100 pmoles probe PT6   4 μLhuman genomic DNA (Promega, G3041)  75 μL water 2.5 units Taq (Promega,M1861)

The PCR cycling parameters were: 94° C., 2 minutes; (94° C., 30 seconds;60° C., 1 minute; 70° C., 1 minutes)×35; 70° C., 7 minutes; 4° C. soak.Ten microliters of the PCR reaction were run on a 1.5% agarose gel,ethidium bromide stained, and a band of correct size was visualizedunder UV light. To 25 μL of the PCR reaction were added 50 units of Exo6, and the sample was incubated at 37° C. for 15 minutes. The sample wasthen treated with Exo 6 as described in Example 38, below, and purifiedaway from the free nucleotides using MagneSil™ paramagnetic particles(Promega, A1330) according to manufacturer's instructions. Fourmicroliters of the 100 μL eluted DNA were interrogated with probes PT7(SEQ ID NO:106), PT8 (SEQ ID NO:107), PT9 (SEQ ID NO:108), and PT10 (SEQID NO:109) in four separate reactions.

PT7 and PT9 differ only in the nucleotide present nine nucleotides fromthe 3′ end. Probe PT7 has a base complementary to the wild type sequencenine bases from the 3′ end, whereas probe PT9 has a mismatching base atthat position. These two probes have a 3′ terminal nucleotide thatmatches wild type prothrombin. PT8 and PT10 differ only in thenucleotide present nine nucleotides from the 3′ end. Probe PT8 has abase complementary to the wild type sequence nine bases from the 3′ end,whereas probe PT10 has a mismatching base at that position. These twoprobes have a 3′-terminal nucleotide that matches the mutantprothrombin, but is a mismatch with wild type.

The interrogation reactions were set up as follows: 4 μL target DNA werecombined with 150 pmol of interrogation oligonucleotide probe (or nonefor control reaction) and water to a final volume of 20 μL. Thesesamples were incubated at 95° C. for 3 minutes, followed by incubationat 37° C. for 10 minutes. Then 20 μL master mix were added, and the tubeincubated at 37° C. for an additional 15 minutes. The master mixcontains 71 μL water, 20 μL 10× DNA Pol buffer (Promega, M195A), 5 μL 40mM NaPPi, 2 μL 10 μM ADP, 1 unit NDPK, and 2 units Klenow exo− (Promega,M218A). Then, 100 μL of L/L reagent (Promega, FF2021) were added and therelative light units measured immediately in a Turner® TD20/20luminometer. The control values from samples lacking an interrogationoligonucleotide were subtracted and the results are reported in theTable below. Interrogation Relative Reaction oligo Light Units 1. PT71520 2. PT8 495 3. PT9 1724 4. PT10 219

The results indicate that the additional mismatch, internally located inthe interrogation probe, helped to increase the level of discriminationobserved between wild type and mutant probes. When the internal mismatchwas not present in the interrogation probes, there was 3.1-folddiscrimination, whereas when the internal mismatch was present in theinterrogation probes, there was 7.9-fold discrimination. PT5 5′ATAGCACTGGGAGCATTGAGGC 3′ SEQ ID NO: 104 PT6 5′ GCACAGACGGCTGTTCTCTT 3′SEQ ID NO: 105 PT7 5′ GTGACTCTCAGCG 3′ SEQ ID NO: 106 PT8 5′GTGACTCTCAGCA 3′ SEQ ID NO: 107 PT9 5′ GTGATTCTCAGCG 3′ SEQ ID NO: 108PT10 5′ GTGATTCTCAGCA 3′ SEQ ID NO: 109

EXAMPLE 31 Determination of the Presence of the Leiden Mutation ofFactor V

A synthetic first nucleic acid target of the Factor V gene was designedto have the wild type sequence that contains a G at position 32 of FV1(SEQ ID NO:25). The complementary strand, FV2, (SEQ ID NO:26) had 4additional bases at its 3′-terminus. A second synthetic nucleic acidtarget of Factor V was designed to have the Leiden mutation, an Aresidue at position 32 of FV3 (SEQ ID NO:27). The mutant complementarystrand, FV4 (SEQ ID NO:28) also had 4 additional bases at its3′-terminus. The nucleic acid target oligonucleotides, FV1 to FV4, wereseparately dissolved at a concentration of one mg/mL in water.

Nucleic acid probe FV5 (SEQ ID NO:29) was synthesized to be totallycomplementary to one strand of the first target, FV1. The probe wassynthesized to place the complementary C residue at an interrogationposition penultimate to the 3′-terminal nucleotide of the probe FV5,corresponding to the G at position 32 of FV1. Similarly, a syntheticnucleic acid probe was prepared having sequence FV6 (SEQ ID NO:30) thatis totally complementary to one strand of the second target, Factor vwith the Leiden mutation, FV3. The probe was synthesized to place thecomplementary T residue at an interrogation position penultimate to the3′-terminal nucleotide of the probe FV6, corresponding to the A atposition 32 of FV3. Nucleic acid probe stock solutions had aconcentration of one mg/mL in water.

The FV1 oligonucleotide was mixed with an equal amount of itscomplementary strand, FV2, heated to 95° C. for about 15 minutes andthen cooled to room temperature to produce a first sample containing adouble stranded DNA segment including the first nucleic acid target,corresponding to the wild type sequence of the Factor V gene.

The FV3 oligonucleotide was mixed with an equal amount of itscomplementary strand having FV4, heated to 95° C. for about 15 minutes,and then cooled to room temperature to produce a second sample thatincluded a double stranded DNA (dsDNA) segment containing the secondtarget, the sequence of the Factor V gene in the region of the Leidenmutation.

One microliter of the dsDNA sample to be assayed for the presence of thefirst or second target was admixed with 1 μL of a nucleic acid probe and18 μL of water to form separate hybridization compositions. Controls had1 μL of the dsDNA sample and 19 μL of water.

They were denatured by heating to 95° C. for three minutes, thenmaintained for 10 minutes under hybridizing conditions (in a 37° C.incubator) to form separate treated samples.

A master mix was assembled containing 10× DNA Polymerase Buffer (20 μL;Promega, M195), sodium pyrophosphate (5 μL of 40 mM Na₄P₂O₇ solution;Promega, C113), Klenow Exo Minus (5 μL; 5U; Promega, M218), NDPK (1 μLof a 10 U/μL solution of NDPK [Sigma, NO379], dissolved in water), ADP(2 μL of a 10 μM solution of ADP [Sigma, A5285] dissolved in water), andwater (67 μL).

The hybridized, treated samples (20 μL) were each mixed with the mastermix and maintained for 15 minutes at 37° C. to form a depolymerizedsample.

The depolymerized sample was added to 100 μL L/L reagent (Promega,F202A), and the amount of light produced was read on a Turner® TD20/20luminometer. A total of 8 samples and two controls were analyzed. Theaveraged results are shown below. Average Assay Nucleic Acid NucleicAcid Relative No. Target in Sample Probe Light Units  1, 2 Factor VFactor V 1063  3, 4 Factor V Factor V Leiden 88.8  5 Factor V none 8.652 6, 7 Factor V Leiden Factor V 139.8  8, 9 Factor V Leiden Factor VLeiden 1016 10 Factor V Leiden none 7.587

The data show that the light signal is about 10 fold greater when thenucleic acid probe is exactly complementary to the nucleic acid target(Assay Nos. 1, 2, 8 and 9) than when the nucleic acid probe is partiallycomplementary to the nucleic acid target with the mismatch at theposition penultimate to the 3′-terminal nucleotide (Assay Nos. 3, 4, 6,and 7). The latter signal is in turn about 10-fold greater than thelight generated when there is no probe to hybridize to the nucleic acidtarget (Assay Nos. 5 and 10). FV1 5′CTAATCTGTAAGAGCAGATCCCTGGACAGGCGAGGAATACAGAGGGCAGCA SEQ ID NO: 25GACATCGAAGAGCT 3′ FV2 5′AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTCGCCTGTCCAGGGATCTG SEQ ID NO: 26CTCTTACAGATTAGAGCT 3′ FV3 5′CTAATCTGTAAGAGCAGATCCCTGGACAGGCAAGGAATACAGAGGGCAGCA SEQ ID NO: 27GACATCGAAGAGCT 3′ FV4 5′AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTTGCCTGTCCAGGGATCTG SEQ ID NO: 28CTCTTACAGATTAGAGCT 3′ FV5 5′ CTGCTGCCCTCTGTATTCCTCG 3′ SEQ ID NO: 29 FV65′ CTGCTGCCCTCTGTATTCCTTG 3′ SEQ ID NO: 30

EXAMPLE 32 Determination of the Presence or Absence of a NucleotideSequence in a Sample Known to be Associated with the Factor V LeidenPhenotype in Humans with Additional Interrogation Probes

In this Example, another pair of probes, complementary to the oppositetemplate strand as those used in Example 31, were used to detect thegene sequences of the wild type Factor V gene and the Leiden allele ofthis gene. Oligonucleotides FV7 (SEQ ID NO:110) and FV8 (SEQ ID NO:111)were dissolved at 1 mg/mL in water. Oligonucleotides FV1 (SEQ ID NO:25),FV2 (SEQ ID NO:26), FV3 (SEQ ID NO:27) and FV4 (SEQ ID NO:28) were usedas targets, and the following solutions were assembled. Target ProbeWater Solution (μL) (μL) (μL)  1 and 2 1 FV1 + FV2 1 FV7 18  3 and 4 1FV1 + FV2 1 FV8 18  5 1 FV1 + FV2 none 19  6 and 7 1 FV3 + FV4 1 FV7 18 8 and 9 1 FV3 + FV4 1 FV8 18 10 1 FV3 + FV4 none 19

The above solutions were heated to 95° C. for three minutes, then placedin a 37° C. incubator for 10 minutes. The following master mix wasassembled: 10 × DNA Polymerase Buffer (Promega M195)  20 μL 40 mM SodiumPyrophosphate (Promega C113)  5 μL 10 U/μl Klenow Exo Minus (PromegaM218)  5 μL NDPK (Sigma, NO379 at 10 U/μL in water)  1 μL ADP (SigmaA5285, 10 μM in water)  2 μL Water  67 μL 100 μL

Twenty microliters of master mix were added to each of the heatednucleotide mixes after incubation at 37° C. for 10 minutes. Theresulting reactions were incubated for 15 minutes at 37° C. and thenadded to 100 μL L/L reagent (Promega, F202A) and the light produced wasimmediately read using a Turner® TD20/20 luminometer.

The following results were obtained. Relative Reaction Light Units 1217.9 2 237.9 3 47.76 4 48.33 5 5.903 6 18.79 7 19.19 8 186.8 9 181.5 105.837

These data show that probe FV7 gave a much stronger signal than FV8 onDNA containing a sequence corresponding the native Factor V gene, andthus can be used to detect this DNA sequence in a sample. Probe FV8 gavea much stronger signal than FV7 on DNA containing a sequence encodingthe Factor V gene in the region of the Leiden mutation. FV1 5′CTAATCTGTAAGAGCAGATCCCTGGACAGGCGAGGAATACAGAGGGCAGCA SEQ ID NO: 25GACATCGAAGAGCT 3′ FV2 5′AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTCGCCTGTCCAGGGATCTG SEQ ID NO: 26CTCTTACAGATTAGAGCT 3′ FV3 5′CTAATCTGTAAGAGCAGATCCCTGGACAGGCAAGGAATACAGAGGGCAGCA SEQ ID NO: 27GACATCGAAGAGCT 3′ FV4 5′AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTTGCCTGTCCAGGGATCTG SEQ ID NO: 28CTCTTACAGATTAGAGCT 3′ FV7 5′ GACAAAATACCTGTATTCCTCG 3′ SEQ ID NO: 110FV8 5′ GACAAAATACCTGTATTCCTTG 3′ SEQ ID NO: 111

EXAMPLE 33 Detection of a Sequence in the Cystic Fibrosis Gene in theRegion of the Delta 508 Mutation

In this Example, an assay was performed to detect a sequence thatencodes a segment of the cystic fibrosis gene spanning the mutationknown as the delta F508 allele.

Oligonucleotides CF1 (SEQ ID NO:112) and CF2 (SEQ ID NO:113) weresynthesized and redissolved in water at a concentration of 50 pmol/μL.These primers were used to produce an amplified segment of the humanchromosomal DNA by PCR amplification. PCR reactions contained 20 nghuman genomic DNA, 50 pmol each primer, 1× Promega Tag Reaction Bufferwith 1.5 mM MgCl2 (Promega, M188A), 200 μM dNTPs, and 1.25 U Tag DNAPolymerase (Promega M186A). Cycling conditions were 1×2 minutes at 94°C., 35×[0.5 minutes at 94° C., 1 minute at 60° C., 1 minute at 72° C.],1×7 minutes at 72° C., 4° C. soak. The amplified DNA was purified usingWizard PCR Preps (Promega A7170) by mixing 25 μL PCR product with 1 mLresin and washing with 3×1 mL 80% isopropanol. This DNA was used torepresent wild type human DNA encoding the cystic fibrosis gene spanningthe delta F508 mutation.

Oligonucleotides CF6 (SEQ ID NO:117) and CF7 (SEQ ID NO:118) weredissolved in water at a concentration of 1 mg/mL, mixed and annealed toform a double strand DNA segment as described for oligonucleotide FV1and FV2 above. This DNA was used to represent human DNA encoding thedelta F508 mutation at this locus.

Oligonucleotide probes CF3 (SEQ ID NO:114), CF4 (SEQ ID NO:115) and CF5(SEQ ID NO:116) were prepared. The sequence of probe CF3 is completelycomplementary to wild type cystic fibrosis gene. The sequence of probeCF4 was identical to that of probe CF3 except for the 3′-terminalnucleotide that is complementary to the nucleotide present in the deltaF508 mutation, and thus was completely complementary to one strand ofthat mutant sequence. The sequence of probe CF5 was completelycomplementary to the second strand of that F508 mutant sequence, andalso therefore differed from a total complement of probe CF3 at the3′-terminal nucleotide. The probes were separately dissolved in water toa concentration of 1 mg/mL. The following solutions were assembled.Probe Target Water Solution (μL) (μL) (μL)  1 and 2 1, CF3 4, Purified,15 Amplified DNA  3 and 4 1, CF4 4, Purified, 15 Amplified DNA  5 and 61, CF5 4, Purified, 15 Amplified DNA  7 none 4, Purified, 16 AmplifiedDNA  8 and 9 1, CF3 1, Annealed 18 (CF6 + CF7) 10 and 11 1, CF4 1,Annealed 18 (CF6 + CF7) 12 and 13 1, CF5, 1, Annealed 18 (CF6 + CF7) 14none 1, Annealed 19 (CF6 + CF7)

These solutions were heated at 95° C. for 3 minutes and then placed in a37° C. incubator for 10 minutes.

A master mix was assembled as described in Example 32 and 20 μL of thissolution were added to each of solutions 1-14 above. They were incubatedfor another 15 minutes at 37° C.

The solutions were added to 100 μL of L/L reagent (Promega F202A)) andthe light produced by the reactions was immediately measured using aTurner® TD 20/20 luminometer. The following results were obtained.Relative Net Solution Light Units Average RLU* 1 1527 762 2 1683 3 766.7(−48.5) 4 823.5 5 893.2 44.1 6 881.0 7 843.4 8 72.73 11.0 9 80.05 10310.9 306.5 11 302.1 12 439.0 434.4 13 429.8 14 65.90*The net value is calculated by averaging the duplicate samples andsubtracting the target alone value measured for the different sets.

These data show that probe CF3 provided much higher signals with wildtype DNA than were provided by either of probes CF4 or CF5 that had amismatched nucleotide at the site of the mutation. In addition, bothprobes CF4 and CF5 that were completely complementary to the mutantsequence provided much higher signals with DNA encoding the delta F508mutant than with wild type DNA. CF1 5′ CATTCACAGTAGCTTACCCA 3′ SEQ IDNO: 112 CF2 5′ GCAGAGTACCTGAAACAGGA 3′ SEQ ID NO: 113 CF3 5′CATCATAGGAAACACCAAG 3′ SEQ ID NO: 114 CF4 5′ CATCATAGGAAACACCAAT 3′ SEQID NO: 115 CF5 5′ GGCACCATTAAAGAAAATATCATT 3′ SEQ ID NO: 116 CF6 5′CTGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATGATGAATATAG SEQ ID NO: 117 CF7 5′CTATATTCATCATAGGAAACACCAATGATATTTTCTTTAATGGTGCC SEQ ID NO: 118 AG 3′

EXAMPLE 34 Detection of a Sequence in the Cystic Fibrosis Gene in theRegion of the Delta 508 Mutation including a Sample Containing Both theNormal and Delta F508 Alleles

This Example demonstrates an assay that detects a sequence encoding asegment of the cystic fibrosis gene spanning the mutation known as thedelta F508 allele. The assay is illustrated using the wild type humansequence of this gene in this region and using a sample that has bothalleles. The results here demonstrate that the assay can discriminatebetween homozygotes for these alleles, and can be used to detectheterozygote samples in which both alleles are present together, aswould be the case with a carrier for a wide variety of genetic diseases.

Oligonucleotides CF8 (SEQ ID NO:119) and CF9 (SEQ ID NO:120), asynthetic wild type target, were dissolved in water and annealed asdescribed for Example 31. CF6 (SEQ ID NO:117) and CF7 (SEQ ID NO:118)were also used as targets. CF3 (SEQ ID NO:114) and CF4 (SEQ ID NO:115)were used as probes. The following solutions were assembled. ProbeTarget(s) Water Solution (μL) (μL) (μL)  1 and 2 1, CF3 1, Annealed 18(CF8 + CF9)  3 and 4 1, CF4 1, Annealed 18 (CF8 + CF9)  5 none 1,Annealed 19 (CF8 + CF9)  6 and 7 1, CF3 1, Annealed 17 (CF6 + CF7) and1, Annealed (CF8 + CF9)  8 and 9 1, CF4 1, Annealed 17 (CF6 + CF7) and1, Annealed (CF8 + CF9) 10 none 1, Annealed 18 (CF6 + CF7) and 1,Annealed (CF8 + CF9) 11 and 12 1, CF3 1, Annealed 18 (CF6 + CF7) 13 and14 1, CF4 1, Annealed 18 (CF6 + CF7) 15 none 1, Annealed 19 (CF6 + CF7)

The above solutions were heated at 95° C. for 3 minutes, then placed ina 37° C. incubator for 10 minutes. A master mix was made as in Example32 and 20 μL of this solution were then added to each of tubes 1-15. Thetubes were incubated at 37° C. for an additional 15 minutes, then thesolutions were added to 100 μL of L/L reagent (Promega F202A) and thelight produced by the reactions was read immediately using a Turner®TD20/20 luminometer. The following data were obtained. Relative AdjustedNet Sample Light Units Light Value* 1 310.1 307.9 2 342.3 3 22.45 4.41 423.02 5 18.29 6 400.2 346.98 7 393.7 8 332.3 269.38 9 306.4 10 49.97 1196.67 37.68 12 109.1 13 371.3 305.4 14 369.8 15 65.22*This value was calculated by averaging the duplicate reactions andsubtracting the value measured for the appropriate target alone controlreaction.

These data again show that probe CF3 provided a much stronger signalwith normal (wild type; homozygous) DNA than did probe CF4, and probeCF4 provided a much stronger signal with the homozygous delta F508mutation target than did probe CF3. In addition, when both targets werepresent in the sample, as in a heterozygote, signals were provided fromboth probes to indicate the presence of a heterozygote. Thus, theanalytical output from this method illustrated whether the nucleic acidtarget sequence in a nucleic acid sample was homozygous or heterozygous,and when homozygous, which of the alleles was present. CF3 5′CATCATAGGAAACACCAAG 3′ SEQ ID NO: 114 CF4 5′ CATCATAGGAAACACCAAT 3′ SEQID NO: 115 CF6 5′ CTGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATGATGAATA TAG 3′SEQ ID NO: 117 CF7 5′ CTATATTCATCATAGGAAACACCAATGATATTTTCTTTAATGGTGCC AG3′ SEQ ID NO: 118 CF8 5′ CTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAG 3′ SEQ ID NO: 119 CF9 5′CTATATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGT GCCAG 3′ SEQ ID NO: 120

EXAMPLE 35 Detection of DNA Sequences Corresponding to the ProthrombinGene in the Region of a Single Nucleotide Polymorphism

An assay for the presence or absence of a mutation in the humanprothrombin gene is illustrated in this Example. This SNP ischaracterized by a G to A substitution in the prothrombin gene.

Oligonucleotides PT1 (SEQ ID NO:121), PT2 (SEQ ID NO:122), PT3 (SEQ IDNO:123), and PT4 (SEQ ID NO:124) were synthesized and dissolved in waterto a concentration of 1 mg/mL. A sample of PT1 and PT2 were then dilutedto 0.3 ng in water for use in the solutions below, and the followingsolutions were made. Probe Target Water Solution (μL) (μL) (μL)  1 and 21, PT3 1, PT1 18  3 and 4 1, PT4 1, PT1 18  5 none 1, PT1 19  6 and 7 1,PT3 1, PT2 18  8 and 9 1, PT4 1, PT2 18 10 none 1, PT2 19

These solutions were heated at 95° C. for three minutes then placed in a37° C. incubator for 10 minutes.

A master mix was assembled as in Example 32 and 20 μL of this solutionwere added to each of solutions 1-10, above, and all solutions wereincubated for 15 minutes at 37° C. After this incubation, thesesolutions were added to 100 μL of L/L reagent (Promega F202A) and thelight produced by the solutions immediately read using a Turner® TD20/20 luminometer. The following results were obtained. Sample RelativeLight Units 1 240.9 2 253.3 3 56.10 4 55.88 5 5.88 6 29.61 7 31.49 8738.0 9 646.8 10 6.21

These data demonstrate the probe PT3 provided much higher signals with awild type target (PT1) than does probe PT4, but that probe PT4 provideda much higher signal with mutant template (PT2) than did PT3. PT1 5′TCCCAATAAAAGTGACTCTCAGCGAGCCTCAATGCTCCCAGTGC TATTCA 3′ SEQ ID NO: 121PT2 5′ TCCCAATAAAAGTGACTCTCAGCAAGCCTCAATGCTCCCAGTGC TATTCA 3′ SEQ ID NO:122 PT3 5′ GGAGCATTGAGGCTCG 3′ SEQ ID NO: 123 PT4 5′ GGAGCATTGAGGCTTG 3′SEQ ID NO: 124

EXAMPLE 36 Detection of DNA Sequences Associated with DNA Translocations

An assay is described in this Example that permits a particular type ofhuman DNA translocation to be detected. The particular translocationtakes place in the region of the bcr gene, and a segment of the abl geneis involved with the translocation.

Oligonucleotides BA1 (SEQ ID NO:125), BA2 (SEQ ID NO:126), BA3 (SEQ IDNO:127) and BA4 (SEQ ID NO:128) were synthesized and dissolved in waterat a concentration of 1 mg/mL. BA1 and BA2 were diluted 1:1000 in waterand the following solutions were assembled. Probe Target Water Solution(μL) (μL) (μL) 1 and 2 1, BA3 1, BA1 18 3 and 4 1, BA4 1, BA1 18  5 none1, BA1 19 6 and 7 1, BA3 1, BA2 18 8 and 9 1, BA4 1, BA2 18 10 none 1,BA2 19

These solutions were heated at 95° C. for 3 minutes then placed in a 37°C. incubator for 10 minutes.

A master mix was assembled as described in Example 32. After solutions1-10 were separately incubated for 10 minutes at 37° C., 20 μL of mastermix were added to each, and the resulting solutions were heated for anadditional 15 minutes at 37° C. After this time, the contents of thetubes were added to 100 μL of L/L reagent (Promega F202A) and theresulting light produced was read immediately using a Turner® TD 20/20luminometer. The following results were obtained. Relative SolutionLight Units 1 1284 2 1414 3 126.9 4 124.7 5 27.21 6 33.73 7 36.68 8 10619 1040 10 24.04

These data show that probe BA3 provided a much greater signal with DNAsequences corresponding to the wild type bcr gene than with mutant DNA.Conversely, probe BA4 provided a much greater signal with a DNA sequencethat corresponds to the sequence from a bcr/abl translocation than withthe normal DNA. BA1 5′ CAGTACTTACTTGAACTCTGCTTAAATCCAGTGGCTGAGT 3′ SEQID NO: 125 BA2 5′ CTGAAGGGCTTTTGAACTCTGCTTAAATCCAGTGGCTGAGT 3′ SEQ IDNO: 126 BA3 5′ TGGATTTAAGCAGAGTTCAAGT 3′ SEQ ID NO: 127 BA4 5′TGGATTTAAGCAGAGTTCAAAA 3′ SEQ ID NO: 128

EXAMPLE 37 Use of Chemical DNA Denaturation of Target DNA Prior toGenotype Determination

In this Example, denaturation of target DNA by chemical agents iscompared to high temperature denaturatin.

An amplified DNA segment containing a segment of the Factor V gene inthe region of the Leiden mutation, but from wild type DNA was producedas described in Example 32.

The amplified DNA was purified using a commercial DNA purificationsystem (Promega, A7170) as described in Example 32. Probe FV7 (SEQ IDNO:110) and FV8 (SEQ ID NO:111) were used, and the following solutionswere assembled. Target* Probe Water Total Solution (μL) (μL) (μL) (μL) 1and 2 4 1, FV7 15 20 150 (pmol) 3 and 4 4 1, FV8 15 20 5 4 none 15 20 6,7, 11, 12, 4 1, FV8 4 9 16, and 17 8, 9, 13, 14, 4 1, FV7 4 9 18 and 1910, 15, and 20 4 none 5 9

Solutions 1-5 were heated at 95° C. for three minutes then put in a 37°C. incubator for 10 minutes. Solutions 6-10 were treated with 1 μL 0.2 Nsodium hydroxide for 1-2 minutes, then 10 μL water were added. Solutions11-15 were treated with 1 μL 0.2 N sodium hydroxide for 1-2 minutes,then 10 μL 50 mM Tris HCl pH 7.3 were added. Solutions 16-20 weretreated with 1 μL 0.2 N sodium hydroxide for 1-2 minutes, then 10 μL 100mM Tris HCl pH 7.3 were added. After these treatments, solutions 7-18were placed in a 37° C. incubator for 5 minutes.

A master mix was assembled as in Example 32 After the treatmentsdescribed above, 20 μL of master mix were added to solutions 1-18 andthe solutions were then incubated for 15 minutes at 37° C. After thisincubation, the contents of the tubes were added to 100 μL L/L reagent(Promega F202A) and the light production from the reactions was readimmediately using a Turner® TD 20/20 luminometer. The following resultswere obtained. Relative Net Average Solution Light Units Light Units 11495 1343 2 1540 3 278.7 100.1 4 269.5 5 174 0 6 625 1383.2 7 1539 8305.1 106.9 9 306.3 10 198.8 0 11 1629 1408.8 12 1638 13 304.9 82.15 14308.8 15 224.7 0 16 1595 1350.2 17 1567 18 303.2 76.95 19 312.3 20 230.80

These data indicate that either chemical denaturation or heatdenaturation can be used prior to primer pyrophosphorylation withoutgreatly affecting the results. FV7 5′ GACAAAATACCTGTATTCCTCG 3′ SEQ IDNO: 110 FV8 5′ GACAAAATACCTGTATTCCTTG 3′ SEQ ID NO: 111

EXAMPLE 38 Reduction of Target Background by Removal of One Strand of byDouble Strand DNA Target

A particular target produced by amplification of a segment of the ricegenome is interrogated in this Example. It was found that this targetproduced high background signal values if nothing is done to eliminateone strand of the amplified DNA target and did not exhibitdiscrimination between two primers that were designed to detect a SNPpresent in some rice strains. This Example illustrates how one canpurposefully destroy one of amplified DNA strands and interrogate theother strand. For this case in particular, such manipulation result ingreatly reduced background light signal from the target, permittingclear determination of the interrogation signal.

Probes RS1 (SEQ ID NO:129) and RS2 (SEQ ID NO:130) were dissolved at aconcentration of 50 pmole/μL in water. Probe RS1 containedphosphorothioate linkages at the first four 5′-terminal linkages thatare not cleaved by the enzyme used in the reaction. DNA was insolatedfrom rice and was at a concentration of 10 μg/mL. The following solutionwas assembled in duplicated: Component Volume (μL) 10 × DNA Polymerasebuffer 5 without MgCl₂ (Promega M190A) 25 mM MgCl₂ (Promega A351A) 3 10mM dNTP mixture (Promega C114A) 1 Primer RS1 (50 pmol/μL) 1 Primer RS2(50 pmol/μL) 1 Rice genomic DNA (10 ng/μL) 1 Water 38 Taq DNA Polymerase(Promega M186A) 1.25 U

These solution were heated to 94° C. for two minutes, then subjected tothe following temperature cycling program for 35 cycles: 0.5 minutes,94° C.; 1 minute, 60° C.; 1 minute, 70° C. Then the solution was held at70° C. for 7 minute then cooled to 4° C.

The two reaction tubes were pooled and mixed and then 13-25 μL sampleswere removed and placed into individual tube. The compositions whitingthe individual tubes were treated with T7 Exonuclease 6 (USB, E700254)as follows. Solution 1 No Exo 6 addition or further heating Solution 250 U of Exo 6 and heated for 15 minutes at 37° C. Solution 3 50 U of Exo6 and heated for 30 minutes at 37° C.

The DNA in the resulting solution was purified using the followingmethod:

-   1. 200 μL of a slurry containing 15 μL MagneSil™ paramagnetic    particle (Promega) in solution containing 0.4 M guanidine    thiocyanate and 0.08 M potassium acetate were added to each sample.-   2. The MagneSil™ paramagnetic particles were mixed in the solution    and held against the side of the tube with a magnet.

3. The particles were washed twice with 200 μL of 70% ethanol byaddiction of the solution to the tubes, resuspension of the particles inthe solution, recapture of the particles against the tube walls with themagnet and removal of the particle-free solution

4. The particles were resuspended in fifty microliters of water.

5. 200 μL 0.4 M GTC and 0.08 M potassium acetate were added to each.

6. Step 2 was repeated as described above except that three washes with70% ethanol were performed.

7. The particle were resuspended in 100 μL water, the particles werecaptured against the side of tube, and the solution containing thepurified DNA was transferred to a new tube

A master mix was made as described in Example 32, and primers RS3 (SEQID NO:131) and RS4 (SEQ ID NO:132) were resuspended at a concentrationof 1 mg/mL in water. Each of the purified DNAs was assembled intoreaction solution as described below. Probe Purified DNA Water Solution(μL) Target (μL) (μL) Wild Type 1, RS3 4 15 (WT) Probe Variant 1, RS4 415 Probe No Probe none 4 16

These solution were heated at 95° C. for 3 minutes, then placed in a 37°C. incubator for 10 minutes. After the 10 minute incubation, 20 μL ofmaster mix were added to all tubes and tubes were incubated again for 15minutes at 37° C. After this second incubation, the solution were addedto 100 μl L/L reagent (Promega, F202A) and the light produced measuredimmediately using a Turner® TD 20/20 luminometer.

The following results were obtained: Relative Light Units MeasuredTarget WT Probe Variant Probe No Probe No Exo 6 759.0 776.0 401.6Treatment 15 min. Exo 6 556.6 138.4 122.3 Treatment 30 min. Exo 6 543.2257.4 203.0 Treatment

Calculation of Net Light Units and Ratio of Response Net Light Units*Target WT Probe Variant Probe Ratio** No Exo 6 357.4 374.4 0.95Treatment 15 min. Exo 6 434.3 16.1 27.0 Treatment 30 min. Exo 6 340.254.4 6.25 Treatment*Net light units are calculated by subtracting the no probe value fromthe other two values**Ratio is calculated by dividing the net light units for the WT probeby the net light units for the variant reaction.

The exonuclease used in this example hydrolyzes double-stranded DNA in a5′ to 3′ direction, but cannot hydrolyze the DNA if phosphorothioatelinkages are present on the 5′ end of the DNA to be digested. Thus, thetreatment used above should eliminate one strand of the amplified DNAmade by extension of primer RS2 but should not eliminate the strand madeby extension of primer RS1. This treatment both reduced the response ofreactions without primer and permitted the discrimination of the SNP atthe interrogation site. RS1 5′ C*C*C*A*ACACCTTACAGAAATTAGC SEQ ID NO:129 3′ RS2 5′ TCTCAAGACACAAATAACTGCAG 3′ SEQ ID NO: 130 RS3 5′AGAACATCTGCAAGG 3′ SEQ ID NO: 131 RS4 5′ AGAACATCTGCAAGT 3′ SEQ ID NO:132*signifies the presence of a phosphorothioate linkage between theindicated bases.

EXAMPLE 39 Determination of SNPs in DNA Isolated from Plant Materials

The procedures detailed in Example 38 are used here to determine thegenotype of rice DNAs at a known SNP site.

Five coded DNA samples and two DNA samples of known genotype (the “G”allele and the “T” allele) were obtained and subjected to amplificationwith probes RS1 (SEQ ID NO:129) and RS2 (SEQ ID NO:130)as described inthe previous Example. The DNA was then treated with T7 Exonuclease 6 for15 minutes at 37° C. and purified as in the previous Example. Theresulting purified DNA was subjected to pyrophosphorylation reactionsusing probes RS3 (SEQ ID NO:131), RS4 (SEQ ID NO:132), or no probe andthe reaction products added to L/L reagent (Promega, F202A) and lightproduction measured as in the previous example.

The following results were obtained: Relative Light Units Measured WTProbe Variant Probe DNA Analyzed (RS3 G Allele) (RS4 T Allele) No Probe#1 784.5 307.5 229.9 #2 286.3 882.7 227.9 #3 291.5 862.4 202.9 #4 560.4195.5 158.2 #5 706.8 235.5 187.7 G Allele 810.7 250.0 189.2 T Allele416.6 1121 243.4

Net Light Units, Ratio and Called Genotype Net Light Units* DNA WTVariant Called Analyzed Probe Probe Ratio** Genotype #1 554.6 77.6 7.1 GAllele #2 58.4 654.8 0.09 T Allele #3 88.6 659.5 0.13 T Allele #4 402.237.3 10.8 G Allele #5 519.1 47.8 10.9 G Allele G Allele 621.5 60.8 10.2G Allele Std. Deviation T Allele 173.2 877.6 0.20 T Allele Std.Deviation*Net light units = total light units − no primer values.**Ratio = Net light units WT primer/net light units variant primer

After these results were obtained, the identity of the DNA samples wasuncoded and all the called genotypes agreed with the previouslydetermined genotype of these samples. These results demonstrate theassay described in this Example can be used to determine SNPs in plantDNA and that removal of one DNA strand of a sample can help eliminatehigh background signals from a template, permitting SNPs to bedetermined. RS1 5′ C*C*C*A*ACACCTTACAGAAATTAGC 3′ SEQ ID NO:129 RS25′ TCTCAAGACACAAATAACTGCAG 3′ SEQ ID NO:130 RS3 5′ AGAACATCTGCAAGG 3′SEQ ID NO:131 RS4 5′ AGAACATCTGCAAGT 3′ SEQ ID NO:132(* signifies the presence of a phosphorothioate linkage between theindicated bases.)

EXAMPLE 40 Improvement in Allele Discrimination by Varying ReactionConditions and ATP Stability in the Pyrophosphorylation Solution afterPyrophosphorolysis

As shown in several previous Examples, pairs of probes can be used todetermine the genotype of a DNA segment using coupled enzymaticreactions. One way to present the discrimination of different allelesusing this technology is to report the relative detection signals as aratio, as shown in the Example above. In this Example, a study isdescribed that illustrates that the ratio between the signals frommatched and mismatched probes can be varied by alteration of thereaction conditions. In addition, the reaction solutions are incubatedon ice to demonstrate that determining the amounts of ATP generatedfollowing pyrophosphorylation of the probes does not have to beperformed immediately if the solutions are placed on ice.

Oligonucleotides CV1 (SEQ ID NO:86), CV2 (SEQ ID NO:35), and CV3 (SEQ IDNO:83) were dissolved to 1 mg/mL in water and CV3 was diluted to aconcentration of 0.3 μg/mL in water. Oligonucleotide CV1 was designed tomatch a known sequence in the CMV viral genome. Oligonucleotide CV2 wasdesigned to match the same region of the viral genome, but to hybridizeexactly to a known drug resistance form of the virus that containedsingle base changes in this region. Oligonucleotide CV3 was designed tomatch a larger region of this viral DNA and is used as a target for thehybridization of probes CV1 and CV2 in the study below.

Nine samples of each of the three following solutions were assembled:Solution 1: 18 μL water, 1 μL CV3 and 1 μL CV1; Solution 2: 18 μL water,1 μL CV2 and 1 μL CV3; and Solution 3: 19 μL water, 1 μL CV3. Thesesolutions were heated at 91° C. for 5 minutes, then cooled at roomtemperature for 10 minutes.

Three solutions of Klenow exo− were prepared by mixing the following:Enzyme solutions Component Klenow #1 Klenow #2 1 × DNA PolymeraseBuffer* 4 μL 8 μL Klenow Exo Minus 6 μL 2 μL (Promega M218B)*= Made by 1:10 dilution of Promega 10 × DNA Polymerase Buffer (M195Awith water).

These manipulations produced solutions of Klenow exo− at concentrationsof 6 U/μL and 2 U/μL for the Klenow #1 and Klenow #2 solutions,respectively.

A master mix was made by assembling the following: Component Amount (μL)10 × DNA Polymerase Buffer 120 water 432 10 mM Sodium Pyrophosphate 15NDPK (1 U/μL) 6 10 μM ADP (Sigma) 12

After mixing, 195 μL samples of master mix were placed into each ofthree separate 1.5 mL microfuge tubes labeled MM#1, MM#2 and MM#3, and 5μL of Klenow exo minus, Klenow #1 and Klenow #2, as described above,were added to those tubes. Twenty microliter samples of each of thosemixes were separately added to each of: 3 tubes of solution 1, threetubes of solution 2 and three tubes of solution 3 after thebefore-described solutions had cooled to room temperature. The tubeswere then incubated at 37° C. for 15 minutes. Four microliters of thesolution in each tube were immediately added to 100 μL of L/L reagent(Promega F202A) and the light production of the resulting solution wasread immediately using a Turner® TD 20/20 luminometer. The tubecontaining the remaining solution was placed on ice. Periodically, fourmicroliter samples of the remaining solution in each of the tubes wereadded to 100 μL of L/L reagent (Promega F120B) and the light productionof the resulting solution read as before to determine if the valuesfirst seen changed over time. The average values for the triplicatereadings are given below. Reading CV1/CV3 CV2/CV3 No Probe Time* (min)Reactions Reactions Reactions Ratio** Klenow used: 5 U/reaction Zero235.4 31.1 2.98 8.26 15 231.9 30.2 2.76 8.40 30 233.8 32.2 3.41 8.00 45219.7 32.8 8.60 8.77 60 218.5 31.8 4.41 7.87 Klenow used: 3 U/reactionZero 200.8 26.3 6.27 9.80 15 min 207.2 31.9 14.9 11.2 30 min 191.3 26.37.30 9.71 45 min 202.0 26.7 6.34 9.62 60 min 192.8 25.9 6.03 9.43 Klenowused: 1 U/reaction Zero 217.5 24.6 8.03 12.65 15 206.8 35.5 23.8 15.9 30200.0 24.4 9.24 12.7 45 210.7 24.9 5.70 10.6 60 210.7 24.5 6.12 11.1*Reading time = time from placement of the tube on ice post 37° C.incubation.**Ratio = ratio obtained by dividing the average net CV1/CV3 reactionvalue by the average net CV2/CV3 reaction value. Net reaction valueswere calculated by subtracting the no probe reaction value from thevalue obtained with the indicated probe.

The ratio calculated above provides a measurement of the relativestrength of the signals of perfectly matched versus mismatched probes atdifferent enzyme levels. Because the ratio is higher as the amount ofenzyme decreased, improved specificity of detection is seen at the lowerenzyme amounts used than at the higher amounts used. Because theabsolute signal strength of the matching probe/target substrate does notvary much over the enzyme levels used, these data illustrate that oneway to improve detection specificity is through the optimization ofenzyme concentrations and that the optimal concentration for the enzymeKlenow exo− can be at or below 1 U/reaction in some cases.

The readings, taken up to an hour after the placement of the reactiontubes on ice, do not show much change versus those read immediately.These results suggest that the ATP level in the reactions does not needto be measured immediately but that the measurement can be performed upto at least one hour post incubation if the solutions are placed on ice.CV1 5′ CACTTTGATATTACACCCATG 3′ SEQ ID NO:85 CV25′ CACTTTGATATTACACCCGTG 3′ SEQ ID NO:35 CV3 5′ GCAACGCTACCTTTGCCATGTTTG3′ SEQ ID NO:83

EXAMPLE 41 Improved Allele Discrimination with Automated ATP Measurement

Improved allele discrimination is demonstrated in this Example byvarying reaction conditions. In addition, the ability to automate thereading of the samples by the use of a plate luminometer that can addthe needed reagent is illustrated.

Oligonucleotides FV1 (SEQ ID NO:25), FV2 (SEQ ID NO:26), FV5 (SEQ IDNO:29) and FV6 (SEQ ID NO:30) were dissolved in water at 1 mg/mL, andthen FV1 and FV2 were diluted to 0.3 μg/mL in water. Solution FV1+2 isassembled from equal volumes of the diluted FV1 and FV2. FV1 and FV2 arecomplementary strands of a segment of the wild type Factor V gene exceptfor a 3′ overhang region of FV2. FV5 is an oligonucleotide probespanning the wild type Factor V gene in the region where the Factor VLeiden mutation occurs in the mutant gene. FV6 is an oligonucleotideprobe spanning the same region as FV5, but it is totally complementaryto the Factor V mutant. Oligonucleotide FV5 is complementary to a regionof FV1 and oligonucleotide FV6 is complementary to FV2. FV5 and FV6 arethe interrogation oligonucleotides.

Six replicates of the solutions below were assembled. FV5 FV6 FV1 + 2Water Solution (μL) (μL) (μL) (μL) Solution #1 1 none 1 18 Solution #2none 1 1 18 Solution #3 none none 1 19 Solution #4 1 none none 19Solution #5 none 1 none 19 Solution #6 none none none 20

These solutions were heated at 91° C. for 7 minutes and cooled at roomtemperature for 15 minutes.

The following master mix was assembled. (μL) 10 × DNA Polymerase Buffer(Promega M195A) 160 40 mM Sodium Pyrophosphate (Promega L350B) 20 NDPK(1 U/μL) 8 10 μM ADP (Sigma) 16 water 556

This solution was mixed and three 253 μL samples were put into separate1.5 mL microfuge tubes. Enzyme dilutions of Klenow exo− toconcentrations of 5 and 2.5 U/μL were made as in Example 40.

A 13.5 μL aliquot of Klenow exo− at 10, 5 and 2.5 U/μL (the lower enzymeconcentrations were produced by dilution of stock enzyme as in theprevious Example) was added to each of the master mix samples. Eachresulting solution was mixed and 20 μL samples of the resultingsolutions were added to two of each of the 6 different solutions heatedat 95° C. before and cooled by incubation at room temperature. Theresulting reaction solutions were heated at 37° C. for 15 minutes andthen placed on ice.

A microtiter plate was taken and 5 μL samples of nanopure water(controls) added to multiple wells and replicate 5 μL samples of thevarious reaction mixes corresponding to the reactions performed at oneenzyme concentration were also added to individual wells in the plate.The plates were then placed on ice. In total, four replicates of twoseparate sets of each reaction were put on the plate. In the same way,two additional plates were assembled with the other reaction mixes forthe other two enzyme concentrations. The plates were then read on aLuminoskan® luminometer that was programmed to add 100 μL of L/L reagent(Promega F202A) and immediately measure the luminescence produced.

In addition, 5 μL samples of the reaction mixes on ice were read intriplicate using a Turner® TD20/20 luminometer by adding the sample to atube containing 100 μL of L/L reagent and immediately reading the lightproduction of the resulting solution.

The averages of the data for the results obtained were calculated andare presented below. Samples labeled ‘match’ contain FV1+2 wild typetarget and FV5 wild type interrogation probe, previously described assolution #1. Samples labeled ‘mismatch’ contain FV1+2 wild type targetand FV6 mutant interrogation probe, previously described as solution #2.Luminoskan Turner TD 20/20 Average Readings Average Readings RelativeRelative Sample Light Units Ratio* Light Units Ratio* 10 U Klenow DataMatch #1 76.55 179.8 Match #2 71.46 210.7 Mismatch#1 3.90 11.16Mismatch#2 4.61 40 11.71 52 Target #1 1.70 5.32 Target #2 1.51 5.16Probe FV1#1 3.89 13.73 Probe FV1#2 3.82 11.75 Probe FV2#1 2.54 7.63Probe FV2#2 2.22 7.06 No DNA#1 1.61 4.37 No DNA#2 1.31 4.90 5 U KlenowData Match #1 59.33 196.0 Match #2 75.59 215.5 Mismatch#1 3.60 11.28Mismatch#2 3.47 54 10.17 43 Target #1 1.77 5.21 Target #2 1.56 4.73Probe FV1#1 3.13 9.89 Probe FV1#2 3.12 9.33 Probe FV2#1 2.19 6.01 ProbeFV2#2 2.22 6.32 No DNA#1 1.58 4.17 No DNA#2 1.46 4.21 2.5 U Klenow DataMatch #1 68.83 235.6 Match #2 72.20 245.8 Mismatch#1 3.08 9.18Mismatch#2 3.33 72 9.83 71 Target #1 1.90 5.11 Target #2 1.65 4.35 ProbeFV1#1 3.11 9.70 Probe FV1#2 2.12 10.99 Probe FV2#1 2.07 5.79 Probe FV2#22.12 6.08 No DNA#1 1.62 4.14 No DNA#2 1.59 4.64*See the text below.

The ratios reported in this Example were determined by first averagingthe results from matching samples then determining the net lightproduction from the matching and mismatching samples and dividing thenet light production from the matching reaction by that seen in themismatch reaction. The net light production was determined bysubtracting the estimated light contribution from the probes and targetpresent in the reactions from the total light produced. The lightproduction from the target reaction was considered to be the total ofthat contributed from the target specifically and that contributed bycontaminating ATP from various components. The net increase from theprobes alone was calculated by subtracting the average No DNA valuesfrom the probe values because it subtracted the contributions fromcontaminating ATP from the probe values. Thus, the formula used todetermine the net light production from the reactions was:Net Light=Total light−[(target alone)+(probe alone−no DNA)]

These data again indicate that improved allele detection results can beobtained by optimizing the amount of enzyme in the reaction. Inaddition, the results indicate that, while reading samples on acommercially available plate luminometer with automated reagent additiondoes not give the same readings as another instrument, the ratio of therelative signal strengths from reactions performed with matched andmismatched probes are approximately equal. Thus, automated reading ofthe reaction products can be used to perform allele determination. FV15′ CTAATCTGTAAGAGCAGATCCCTGGACAGGCGAG SEQ ID NO:25GAATACAGAGGGCAGCAGACAT 3′ FV2 5′ ATGTCTGCTGCCCTCTGTATTCCTCGCCTGTCCA SEDID NO:26 GGGATCTGCTCTTACAGATTAG 3′ FV5 5′ CTGCTGCCCTCTGTATTCCTCG 3′ SEQID NO:29 FV6 5′ CTGCTGCCCTCTGTATTCCTTG 3′ SEQ ID NO:30

EXAMPLE 42 The Effects of Enzyme Reduction on Allele Discrimination

Allele discrimination is further improved in this Example by furtherreduction in the amount of enzyme used in the pyrophosphorylationreaction. Oligonucleotides CM1 (SEQ ID NO:83), CM2 (SEQ ID NO:35) andCM3 (SEQ ID NO:86) were dissolved in water, and CM1 was then diluted to0.3 μg/mL in water. These reagents were used to form the followingsolutions. Solution CM1 (μL) CM2 (μL) CM3 (μL) Water (μL) #1 1 1 — 18 #21 — 1 18 #3 1 — — 19 #4 — 1 — 19 #5 — — 1 19 #6 — — — 20

These solutions were heated to 91° C. for 7 minutes, then permitted tocool for 15 minutes at room temperature. A master mix was assembled asin Example 41 except that the volumes used permitted 5-390 μL samples ofthe final mix to be placed into 5 0.5 mL microfuge tubes.

Klenow Exo− was diluted as described in Example 40 except the dilutionswere adjusted so the diluted solutions contained 20, 10, 5, and 2.5units of enzyme per 10 μL of diluted material. Ten microliters of thediluted enzyme solutions were added to four tubes containing 390 μL ofmaster mix. Ten microliters of 1× DNA polymerase buffer (made by 1:10dilution of 10× DNA Polymerase buffer with water) were added to a fifthtube with master mix to serve as a no-enzyme control. Twenty microlitersof each of the master mixes and control mix were added to groups ofthree of solutions 1-6 above and each resulting solution was heated at37° C. for 15 minutes, then the tube was placed on ice. After all tubeswere processed in this manner, 5 μL samples from each of the tubes wereseparately added to 100 μL of L/L reagent (Promega, F202A) and the lightproduced by the solution immediately read using a Turner® TD 20/20luminometer. The results for the triplicate determinations were averagedand those averages are presented in the table below. Relative LightUnits Measured in Reactions at Various Enzyme Levels (enz/rx) Reaction 2U 1 U 0.5 U 0.25 U Type enz/rx enz/rx enz/rx enz/rx no enz Matching279.0 301.3 306.6 268.3 3.94 probe Mismatched 24.51 23.95 25.02 13.704.35 probe CM1 alone 4.33 4.55 4.75 4.42 3.71 CM2 alone 3.90 4.05 3.914.44 3.75 CM3 alone 7.06 5.06 4.13 4.57 3.93 No DNA 3.35 3.52 4.01 3.943.27 Ratio* 16.6 16.6 15.0 30.4 (nd)*Ratio determined by taking the net relative light units as described inExample 41 for the matching and mismatched primers and dividing thematching probe value by the mismatched probe value.(nd) = not determined.

These data show that a very large degree of allele discrimination can beobtained by lowering the Klenow Exo− level to 0.25 U/reaction. CM15′ GCAACGCTACCTTTGCCATGTTTG 3′ SEQ ID NO:83 CM2 5′ CACTTTGATATTACACCCGTG3′ SEQ ID NO:35 CM3 5′ CACTTTGATATTACACCCATG 3′ SEQ ID NO:86

EXAMPLE 43 Reduction of Background Light Production by Reduction ofEnzyme Concentrations

A method to reduce the signal production from probes is demonstrated inthis Example. Thus, probes PH1 (SEQ ID NO:1), PH2 (SEQ ID NO:2), PH3(SEQ ID NO:133), and PH4 (SEQ ID NO:3) were dissolved in water to aconcentration of 1 mg/mL. The following solutions were assembled induplicate. Solution PH Probe (μL) Water (μL) #1 — 20 #2 1, PH1 19 #3 1,PH2 19 #4 1, PH3 19 #5 1, PH4 19

The solutions were heated at 95° C. for 5 minutes then cooled at roomtemperature for 10 minutes. The following two master mixes wereassembled. 0.25 U Master 5.0 U Master Component Mix (μL) Mix (μL) 10 ×DNA Polymerase 20 20 Buffer Klenow Exo− (1 U/μL)* 1.25 — Klenow exo− (10U/μL) — 2.5 40 mM Sodium 2.5 2.5 Pyrophosphate NDPK (1 U/μL) 1 1 ADP (10μM, Sigma) 2 2 Water 73.25 72 100 100*Made by a 1:10 dilution of Klenow exo− with 1 × DNA polymerase buffer(1 × DNA Polymerase buffer made by 1:10 dilution of 10 × DNA PolymeraseBuffer).

These master mixes were mixed and 20 μL of each master mix were added toone of each of solutions 1-5 above and heated at 37° C. for 15 minutes.Duplicate four microliter samples of each solution containing DNA wereadded to 10 μL L/L reagent (Promega F202A) and the light produced wasimmediately read using a Turner® TD 20/20 luminometer. A single 4 μLsample of the reactions not containing DNA was also read by adding it to100 μl of L/L reagent and reading as above. The following results wereobtained. Relative Light Units 0.25 U Master Mix 5.0 U Master MixReactions (μL)* Reactions (μL)* Solution 1^(st). 2^(nd). Avg. 1^(st).2^(nd). Avg. #1 (no DNA) 6.89 7.32 #2 (PH1) 6.82 6.36 6.60 8.42 8.638.50 #3 (PH2) 17.38 14.25 15.8 195.1 185.8 190.3 #4 (PH3) 20.4 20.4 20.4256.6 381.0 318.8 #5 (PH4) 8.35 7.56 7.96 20.24 32.68 26.5*Data are from a first (1^(st).) and second (2^(nd).) reading that areaveraged (Avg.).

These data indicate that probes PH2 and PH3 produce very highprobe-alone light signals when a master mix containing 5 U of Klenowexo−/reaction was used, and produced a greatly reduced light signal when0.25 U of Klenow exo−/reaction was used. Thus, some probes that producevery high light values with one enzyme concentration can be useful inallele determination reactions if used in reactions with a loweredamount of enzyme. PH1 5′ CTGAACATGCCTGCCAAAGACG 3′ SEQ ID NO:1 PH25′ CTGAACATGCCTGCCAAAGATG 3′ SEQ ID NO:2 PH3 5′ CAGGAACGTAGGTCGGACACGT3′ SEQ ID NO:133 PH4 5′ CAGGAACGTAGGTCGGACACAT 3′ SEQ ID NO:3

EXAMPLE 44 Discrimination of Repeated DNA Sequences UsingPyrophosphorylation-Based Assay Methods

This Example illustrates an assay for determining the number of repeatsof a four base pair sequence in a DNA. Discrimination of such repeatsequences has been found to be very useful for identification offorensic samples. The probes in this set, TR1 (SEQ ID NO:137), TR2 (SEQID NO:138) and TR3 (SEQ ID NO:139) were designed to exactly match knownalleles of the THO 1 locus with 6, 7, and 8 repeats respectively, of aCATT sequence.

Probes TR1-TR3 were suspended in water to a concentration of 1 mg/mL.Targets that were homozygous for THO 1 alleles with 6, 7 and 8 repeatswere amplified using the protocol in the Gene Print™ System instructions(Promega). These targets were named allele 6 (SEQ ID NO:134), allele 7(SEQ ID NO:135) and allele 8 (SEQ ID NO:136), respectively. Gel-purifiedtargets were PCR amplified and then further purified using the Wizard™PCR Clean-up system (Promega, A7170) and the concentration of the DNAmeasured by DNAQuant (Promega). These targets were adjusted to aconcentration of 1 μg/mL and to 3.3 μg/mL by the addition of deionizedwater. The following solutions containing probes were assembled in afinal volume of 20 μL by the addition of water. Solution Probe Target(ng) #1 — allele 6, 1 #2 TR1 allele 6, 1 #3 TR2 allele 6, 1 #4 TR3allele 6, 1 #5 — allele 7, 1 #6 TR1 allele 7, 1 #7 TR2 allele 7, 1 #8TR3 allele 7, 1 #9 — allele 8, 1 #10 TR1 allele 8, 1 #11 TR2 allele 8, 1#12 TR3 allele 8, 1 #13 — allele 6, 3.3 #14 TR1 allele 6, 3.3 #15 TR2allele 6, 3.3 #16 TR3 allele 6, 3.3 #17 — allele 7, 3.3 #18 TR1 allele7, 3.3 #19 TR2 allele 7, 3.3 #20 TR3 allele 7, 3.3 #21 — allele 8, 3.3#22 TR1 allele 8, 3.3 #23 TR2 allele 8, 3.3 #24 TR3 allele 8, 3.3

These solutions were heated at 95° C. for 3 minutes, then permitted tocool by incubation at room temperature for 10 minutes.

The following master mix was made. Amount Component (μL)/reaction 10 ×DNA Polymerase Buffer 4 40 mM Sodium Pyrophosphate 0.5 10 μM ADP 0.4Klenow exo−, 10 U/μL 0.5 NDPK, 1 U/μL 0.2 Nanopure water 14.4

This solution was mixed and 20 μL of this solution were added tosolutions 1-24 above, and the resulting solutions incubated for 15minutes at 37° C. After this incubation, 4 μL of the resulting solutionwere added to 100 μL of L/L reagent(Promega, F202A) and the lightproduced was immediately measured using a Turner® TD20/20 luminometer.The following data were obtained. Relative Solution Light Units #1 3.97#2 50.79 #3 5.94 #4 6.03 #4 3.79 #5 67.23 #7 28.94 #8 6.73 #9 3.19 #1049.52 #11 30.99 #12 30.63 #13 8.62 #14 256.90 #15 16.74 #16 13.83 #176.73 #18 206.5 #19 110.2 #20 15.35 #21 6.49 #22 271.9 #23 150.5 #24154.8

The values from the no probe reactions above were subtracted from thevalues for the various probe/target matches and the resulting values areshown in the table below. Relative Light Units With 1 ng of TargetAllele TR1 TR2 TR3 Assayed Probe Probe Probe Allele 6 44.46 −0.01 −0.44Allele 7 61.07 23.17 0.44 Allele 8 43.97 25.83 24.95

Relative Light Units With 3.3 ng of Target Allele TR1 TR2 TR3 AssayedProbe Probe Probe Allele 6 245.08 4.88 1.73 Allele 7 196.58 100.24 5.14Allele 8 262.22 140.78 144.83

If, in the repeat region, the probe contains the same number of repeatsas the target or fewer, no mismatching bases should be present at the 3′end of the probe and a relatively strong signal is obtained. The TR1probe shows such a signal with targets containing 6, 7 or 8 repeats.However, if the probe contains more repeats in this region than arepresent in the target, mismatched bases are expected at the 3′ end ofthe probe that should greatly reduce the signal developed. As expected,the TR3 probe gave a strong signal against the allele 8 target, but gavea much weaker signal against the allele 7 and 6 targets. Because thesignal generated using the various probes can be used to determine thenumber of repeated units in the repeat region, this method can be usedto determine the alleles present in samples.

Using the above method, probes containing the same number or fewerrepeated sequences as the target produced similar light output. Whenmore repeats were present in the probe than the target, low analyticaloutputs were observed. The number of repeats in the target could thusaccurately be determined by an indicative change in the analyticaloutput, here, luminescence, between the separately assayed samples.Allele 6 5′ GGTGAATGAATGAATGAATGAATGAATGAGGGAA SEQ ID NO:134ATAAGGGAGGAAGAGGCCAATGGG 3′ Allele 75′ GGTGAATGAATGAATGAATGAATGAATGAATGAG SEQ ID NO:135GGAAATAAGGGAGGAAGAGGCCAATGGG 3′ Allele 85′ GGTAGGTGAATGAATGAATGAATGAATGAATGAA SEQ ID NO:136TGAATGAGGGAAATAAGGGAGGAAGAGGCCAATGG G 3′ TR15′ CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATT SEQ ID NO:137CATTCATTCATTCATTCATTCACC 3′ TR2 5′ CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTSEQ ID NO:138 CATTCATTCATTCATTCATTCATTCACC 3′ TR35′ CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATT SEQ ID NO:139CATTCATTCATTCATTCATTCATTCATTCACC 3′

EXAMPLE 45 Discrimination of Repeated DNA Sequences UsingPyrophosphorylation Based Assay Methods Using Another Class of Probes

A surprising result is presented in this Example that demonstrates thata class of probes that should only produce signals with targets of acertain class, essentially give equivalent signals with additionaltargets. Although these results do not match the predicted results, theycan still be used to determine the allelic composition of samples.

The probes in the Example above were designed to hybridize to alleles ofTHO 1 that are used for genotyping humans. They were designed tohybridize as illustratively shown below for probe TR2 with targets ofthree alleles.

Hybridization of probe TR2 (top strand) with an Allele 6 target (bottomstrand): CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT (CATT)₄CATTCATTCACCGGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA (GTAA)₄GTAAGTGG

Hybridization of probe TR2 (top strand) with an Allele 7 target (bottomstrand): CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT (CATT)₄CATTCATTCACCGGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA (GTAA)₄GTAAGTAAGTGG

Hybridization of probe TR2 (top strand) with an Allele 8 target (bottomstrand): CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT (CATT)₄CATTCATTCACCGGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA (GTAA)₄GTAAGTAAGTAAGTGGATGG 5′

As described in Example 44, when the target contains fewer repeats thanthe probe, mismatched bases can occur at the 3′ end of the probe,creating a double strand DNA region that is a very poor substrate forthe pyrophosphorylation reaction. These predictions were verified withthe results obtained.

In this Example, a new form of probe is used in the reactions. Theseprobes are designed to extend beyond the repeat region and hybridize tothe target following this DNA segment when they are hybridized to theallele with the correct number of repeat segments. The predictedhybridization segments for the allele 7 probe (TR6) with the allele 6,7, and 8 targets are shown below.

Hybridization of probe TR6 (top strand) with an Allele 6 target (bottomstrand): CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT (CATT)₄CATTCATTCACCGGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA (GTAA)₄GTAAGTGGATGG

Hybridization of probe TR6 (top strand) with an Allele 7 target (bottomstrand): CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT (CATT)₄CATTCATTCACCGGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA (GTAA)₄GTAAGTAAGTGGA TGG 5′

Hybridization of probe TR6 (top strand) with an Allele 8 target (bottomstrand): CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT (CATT)₄CATTCATTCACCGGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA (GTAA)₄GTAAGTAAGTAAT GGAGTGG 5′

As shown above, probe TR6 was designed to form a product without 3′ endmismatches with only allele 7. Thus, this probe was expected to onlygive a strong signal with the allele 7 target.

In order to test the actual signals that such probes would give withvarious targets, probes TR4 (SEQ ID NO:140), TR5 (SEQ ID NO:141), TR6(SEQ ID NO:142), TR7 (SEQ ID NO:143) and TR8 (SEQ ID NO:144) weredissolved in water to a concentration of 1 mg/mL. These probes were usedwith the targets Allele 6 (SEQ ID NO:134), Allele 7 (SEQ ID NO:135) andAllele 8 (SEQ ID NO:136) to generate the following solutions. As in theExample above, the final volume of these solutions was adjusted to 20 μLby the addition of water. The probes were used at a concentration of 1μg/reaction. Soln. Probe Target (ng) #1 — Allele 6, 1 #2 TR4 Allele 6, 1#3 TR5 Allele 6, 1 #4 TR6 Allele 6, 1 #5 TR7 Allele 6, 1 #6 TR8 Allele6, 1 #7 — Allele 7, 1 #8 TR4 Allele 7, 1 #9 TR5 Allele 7, 1 #10 TR6Allele 7, 1 #11 TR7 Allele 7, 1 #12 TR8 Allele 7, 1 #13 — Allele 8, 1#14 TR4 Allele 8, 1 #15 TR5 Allele 8, 1 #16 TR6 Allele 8, 1 #17 TR7Allele 8, 1 #18 TR8 Allele 8, 1 #19 — Allele 6, 3.3 #20 TR4 Allele 6,3.3 #21 TR5 Allele 6, 3.3 #22 TR6 Allele 6, 3.3 #23 TR7 Allele 6, 3.3#24 TR8 Allele 6, 3.3 #25 — Allele 7, 3.3 #26 TR4 Allele 7, 3.3 #27 TR5Allele 7, 3.3 #28 TR6 Allele 7, 3.3 #29 TR7 Allele 7, 3.3 #30 TR8 Allele7, 3.3 #31 — Allele 8, 3.3 #32 TR4 Allele 8, 3.3 #33 TR5 Allele 8, 3.3#34 TR6 Allele 8, 3.3 #35 TR7 Allele 8, 3.3 #36 TR8 Allele 8, 3.3

These solutions were heated at 95° C. for 3 minutes, then cooled at roomtemperature for 10 minutes. A master mix was assembled and added tothese solutions as in the previous Example. The resulting solutions werethen heated at 37° C. for 15 minutes and were sampled as in the previousExample. The samples were added to L/L reagent (Promega, F202A) and thelight output was immediately measured as in the previous Example. Thefollowing results were obtained.

Relative Light Units From Reactions Containing Probes With 1 ng ofTarget Target — TR4 TR5 TR6 TR7 TR8 Allele 6 2.14 47.40 33.87 11.45 7.577.98 Allele 7 2.06 53.00 30.97 30.43 12.38 10.41 Allele 8 2.51 21.3027.54 30.99 39.04 14.84 (none) (nd) 2.28 2.30 2.59 3.12 3.43 Expected —A 5 A 6 A 7 A 8 A 9 Allele Detected

Relative Light Units from Reactions Containing Probes With 3.3 ng ofTarget Target — TR4 TR5 TR6 TR7 TR8 Allele 6 8.52 282.6 291.5 90.6261.34 46.49 Allele 7 12.23 276.2 237.8 286.4 103.4 74.92 Allele 8 10.33170.6 242.5 264.4 264.9 111.9 (none) (nd) 3.56 3.11 3.25 3.63 3.60Expected — A 5 A 6 A 7 A 8 A 9 Allele Detected

Surprisingly, these probes did not provide the expected detectionpattern. For example, probe TR6 was expected to only give a strongsignal with a target with allele 7 (A 7). Although the probe did show alower signal with allele 6 (A 6) than with allele 7 (90.6 vs. 286.4units, respectively), very little difference was seen between thesignals with alleles 7 and 8 (A 8) (286.4 vs. 264.4 units respectively).In general, all the probes exhibited substantially equal reactivity withany target that had the same number of repeated units or greater thanthe number of repeated units in the probe. These same probes showedlower signals with targets having fewer repeat units than those presentin the probe, with the signal strength seen decreasing as the differencein the number of repeat units increased. Thus, although these probesclearly did not provide the expected signal patterns, they can be usedto determine THO 1 alleles. Allele 6 5′GGTGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAGGAAGAGGC SEQ ID NO: 134CAATGGG 3′ Allele 7 5′GGTGAATGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAGGAAG SEQ ID NO: 135AGGCCAATGGG 3′ Allele 8 5′GGTAGGTGAATGAATGAATGAATGAATGAATGAATGAATGAGGGAAATAAG SEQ ID NO: 136GGAGGAAGAGGCCAATGGG 3′ TR4 5′ CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTSEQ ID NO: 140 CATTCATTCACC 3′ TR5 5′CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTCATT SEQ ID NO: 141CATTCATTCACC 3′ TR6 5′ CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTCATTSEQ ID NO: 142 CATTCATTCATTCACC 3′ TR7 5′CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTCATT SEQ ID NO: 143CATTCATTCATTCATTCACC 3′ TR8 5′CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTCATT SEQ ID NO: 144CATTCATTCATTCATTCATTCACC 3′

EXAMPLE 46 Additional Probes for Detection of THO 1 Alleles

Additional probes are used in this Example to demonstrate that thecreation of additional mismatches between THO 1 allele targets andprobes can result in the formation of probe/target combinations thatprovide strong signals with essentially one THO 1 allele.

Probes TR 9 (SEQ ID NO:145), TR10 (SEQ ID NO:146) and TR11 (SEQ IDNO:147) were dissolved at 1 mg/mL and assembled into reactions withtarget at 3 ng/reaction with allele 6 (SEQ ID NO:134), allele 7 (SEQ IDNO:135), and allele 8 (SEQ ID NO:136) of THO 1 and without any target asdescribed in the Example above. These solutions were heated and cooledas in the previous Example. The resulting solutions were treated withmaster mix, incubated, added to L/L reagent (Promega, F202A) and thelight produced measured as in the previous Example. The followingresults were obtained. Relative Light Units Probe Probe Probe Target TR9TR10 TR11 none Allele 6 59.74 35.86 75.78 8.96 Allele 7 51.73 2.32 15.8510.54 Allele 8 58.58 25.37 33.67 9.85 (none) 47.27 34.24 3.676 (nd)

The values for the probe alone and target alone reactions weresubtracted from the values for the combined reactions and are shown inthe table below. Relative Light Units Target Probe TR9 Probe TR10 ProbeTR11 Allele 6 3.51 −7.34 63.14 Allele 7 −6.08 −22.46 1.63 Allele 8 1.46−18.72 20.14

Increasing the number of mismatched bases between the probe and targetlowers the signal value measured, and in many cases decreases the valuesseen below those attributable from background reactions. In particular,probes TR9, which has a mismatch of 2 base pairs, and TR10, which has anA to C mutation 3 bases from the end of the probe, do not exhibit theability to detect THO 1 alleles. However, probe TR11, which has a A to Gchange 3 bases from the end of the probe, produced a measurable signalwith the allele 6 target that is greater than the signals seen with theother targets.

Probes TR12 (SEQ ID NO:148) and TR13 (SEQ ID NO:149) were then used asabove. The following data were obtained. Relative Light Units TargetProbe TR12 Probe TR13 Allele 6 9.7 9.8 Allele 7 5.0 7.1 Allele 8 10.412.6 (none) 3.0 2.9

These probes, having additional mismatches four base pairs from the 3′end of the probe, only provided very low light signals and apparentlydid not discriminate between the alleles of THO 1. Thus, these datasuggest that probes that can provide allele-specific signals can beidentified by designing probes with base pair mismatches placed in theprobe sequence near the 3′ end of the probe. Allele 6 5′GGTGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAGGAAGAGGC SEQ ID NO: 134CAATGGG 3′ Allele 7 5′GGTGAATGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAGGAAG SEQ ID NO: 135AGGCCAATGGG 3′ Allele 8 5′GGTAGGTGAATGAATGAATGAATGAATGAATGAATGAATGAGGGAAATAAG SEQ ID NO: 136GGAGGAAGAGGCCAATGGG 3′ TR9 5′ CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTSEQ ID NO: 145 CATTCATTCATTCAGC 3′ TR10 5′CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO: 146CATTCATTCATTCCCC 3′ TR11 5′ CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTSEQ ID NO: 147 CATTCATTCATTCGCC 3′ TR12 5′CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO: 148CATTCATTCATTGACC 3′ TR13 5′ CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTSEQ ID NO: 149 CATTCATTCATTAACC 3′

EXAMPLE 47 Discrimination of Repeated DNA Sequences UsingPyrophosphorylation-Based Assay Methods-III

In this Example, PCR targets spanning between 6 to 13 copies of the TPOXfour nucleotide short tandem repeat were discriminated with probesspecific for the number of repeats using a pyrophosphorylation basedassay. The targets were prepared by standard PCR amplification of eachof the (Promega, DC5111) TPOX bands that were previously gel-purified.The PCR cycling parameters were 94° C., 1 minute (94° C., 15 seconds;60° C., 30 seconds; 72° C., 60 seconds)×35, 68° C., 10 minutes. Thecorrect size of the PCR products was confirmed on a 4% polyacrylamidegel electrophoresis. The PCR products were purified using Wizard® PCRPurification System (Promega, A7170) and resuspended in water to aconcentration of 10 ng/μL. The interrogation sequence probes were P6(SEQ ID NO:150), P7 (SEQ ID NO:151), P8 (SEQ ID NO:152), P9 (SEQ IDNO:153), P10 (SEQ ID NO:154), P11 (SEQ ID NO:155), P12 (SEQ ID NO:156)and P13 (SEQ ID NO:157).

Targets containing between 6 and 13 TGAA repeats were each interrogatedwith each of the interrogation probes listed above. The target allelesused were A6 (SEQ ID NO:158), A7 (SEQ ID NO:159), A8 (SEQ ID NO:160), A9(SEQ ID NO:161), A10 (SEQ ID NO:162), A11 (SEQ ID NO:163), A12 (SEQ IDNO:164) and A13 (SEQ ID NO:165), respectively. The probes were at afinal concentration of 2.5 μM in the solution, 10 ng of target were usedper reaction and the volume was increased to 20 μL with water. Thesolutions were heated at 95° C. for 2 minutes, then cooled at roomtemperature over 10 minutes.

Twenty microliters of master mix were added to each solution (14.7 μLwater, 4 μL 10× DNA polymerization buffer, 5 μL 40 mM NaPPi, 0.4 μL 10μM ADP, 0.2 μL NDPK (1 U/μL), 0.2 μL Klenow exo− (10 U/μL)) and theywere further incubated at 37° C. for 15 minutes. Then, 4 μL of thesolution were added to 100 μL of L/L reagent and the light output readwith a Turner® TD20/20 luminometer. The relative light units (rlu)obtained are reported below: Raw rlu numbers Probe Target P6 P7 P8 P9P10 P11 P12 P13 None A6 57.62 16.80 15.65 28.51 23.37 25.31 26.70 47.4814.08 A7 23.29 73.44 22.39 37.28 20.31 25.01 26.29 44.24 25.06 A8 25.0420.82 54.63 35.78 20.69 21.02 22.99 37.51 18.37 A9 28.83 21.60 25.0385.98 30.71 28.55 29.54 50.86 21.48 A10 27.69 25.53 30.30 42.38 61.0430.21 27.71 46.30 32.80 A11 30.29 35.07 30.67 51.20 40.37 69.92 39.1258.52 30.00 A12 35.36 25.43 29.71 45.14 28.44 38.76 63.31 57.05 40.24A13 39.35 27.67 29.92 42.56 33.59 32.80 36.04 84.37 32.70 None 8.67 6.299.15 27.98 14.18 16.51 16.22 32.98 4.66

The above values were adjusted for background and the negative numbersconverted to zero to provide the data in the table below. Probe TargetP6 P7 P8 P9 P10 P11 P12 P13 A6 40 1 0 0 0 0 1 5 A7 0 47 0 0 0 0 0 0 A8 31 32 0 0 0 0 0 A9 3 0 0 41 0 0 0 1 A10 0 0 0 0 19 0 0 0 A11 0 3 0 0 1 280 0 A12 0 0 0 0 0 0 12 0 A13 3 0 0 0 0 0 0 23

The data indicate that the interrogation probes can recognize thepresence of the related homozygote alleles of the TPOX locus. Similarly,heterozygote targets were assayed with the same set of interrogationprobes. Ten nanograms of each purified PCR target were included in eachinterrogation reaction. The reaction conditions were identical to thosefor the homozygote targets described above. The rlu values obtained arereported below. Raw rlu numbers Probe Target P6 P7 P8 P9 P10 P11 P12 P13None A6, 91.09 36.85 40.90 50.88 41.93 41.33 83.69 83.69 31.78 A13 A11,55.94 48.13 54.35 62.64 50.92 84.19 80.33 66.41 54.62 A12 A9, 61.7543.49 47.71 93.76 77.91 37.18 39.11 59.74 33.37 A10 A7, A8 35.66 76.0462.03 36.03 27.92 30.07 31.92 51.40 50.52 None 6.29 4.87 6.22 20.8310.89 11.86 12.41 28.64 4.32

The values were adjusted for background and the negative numbersconverted to zero to provide the data in the table below. Probe TargetP6 P7 P8 P9 P10 P11 P12 P13 A6, A13 57.35 4.53 7.23 2.59 3.58 2.01 6.4527.59 A11, A12 0 0 0 0 0 22.03 17.62 0 A9, A10 26.42 9.58 12.45 43.8837.97 0 0 2.05 A7, A8 0 24.98 9.62 0 0 0 0 0

As can be seen from the results in the table above, this methodaccurately identified each of the heterozygote targets, although probeP6 also identified one false positive for an unknown reason.

Interrogation Probe Sequences: P6 5′ GGCACTTAGGGAACCCTCAC TGAA SEQ IDNO: 150 TGAA TGAA TGAA TGAA TGAA TATT 3′ P7 5′ GGCACTTAGGGAACCCTCAC TGAASEQ ID NO: 151 TGAA TGAA TGAA TGAA TGAA TGAA TATT 3′ P8 5′GGCACTTAGGGAACCCTCAC TGAA SEQ ID NO: 152 TGAA TGAA TGAA TGAA TGAA TGAATGAA TATT 3′ P9 5′ GGCACTTAGGGAACCCTCAC TGAA SEQ ID NO: 153 TGAA TGAATGAA TGAA TGAA TGAA TGAA TGAA TATT 3′ P10 5′ GCACTTAGGGAACCCTCAC TGAASEQ ID NO: 154 TGAA TGAA TGAA GAA TGAA TGAA TGAA TGAA TGAA TATT 3′ P115′ GGCACTTAGGGAACCCTCAC TGAA SEQ ID NO: 155 TGAA TGAA TGAA TGAA TGAATGAA TGAA TGAA TGAA TGAA TATT 3′ P12 5′ GGCACTTAGGGAACCCTCAC TGAA SEQ IDNO: 156 TGAA TGAA TGAA TGAA TGAA TGAA TGAA TGAA TGAA TGAA TGAA TATT 3′P13 5′ GGCACTTAGGGAACCCTCAC TGAA SEQ ID NO: 157 TGAA TGAA TGAA TGAA TGAATGAA TGAA TGAA TGAA TGAA TGAA TGAA TATT 3′

Target Alleles: A6: 5′ GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ IDNO: 158 ATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3′ A7:5′ GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO: 159ATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3′ A8:5′ GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO: 160ATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3′ A9:5′ GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO: 161ATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3′ A10:5′ GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO: 162ATGAATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3′ A11:5′ GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO: 163ATGAATGAATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3′ A12:5′ GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO: 164ATGAATGAATGAATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3′A13: 5 GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO: 165ATGAATGAATGAATGAATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGG ACAGTAGAGTCAACCTCA3′

EXAMPLE 48 Interrogation for Loss of Heterozygosity

In certain types of disease states such as some cancers, there is achange in the heterozygosity of the locus of certain alleles. Forexample, a non-cancerous cell may be heterozygous at a particular locus.In a cancer cell, however, one of the two alleles may be lost or deletedat the particular locus. This is referred to as loss of heterozygosity.

This type of loss of heterozygosity (LOH) reaction was created byPCR-amplifying 25 ng (1 μL) and 50 ng (2 μL) of two E. Coli targets(W3110, DH5α) with probes 10730 (SEQ ID NO:166) and 10731 (SEQ IDNO:167) as described below. These probes span the ΔM15 93 bp deletionpresent in DH5α DNA, but not present in W3110 DNA. The number of PCRcycles was optimized so amplification of the “heterozygote” target (1 μLW3110 and 1 μL DH5α) produced one-half the amount of DNA in each band asdid amplification of the “homozygote” target (2 μL W3110 or 2 μL DH5α)under the same amplification conditions.

PCR targets spanning the locus of interest were created in duplicate asfollows:

2 μL E. coli genomic DNA, W3110 or DH5α for homozygote sample (50 ng); 1μL each W3110 and DH5α for the heterozygote sample; 1 μL of W3110 orDH5α for the LOH sample.   5 μl 10 × Taq buffer with 15 mM MgCl₂(Promega, M188A) 0.5 μL Probe 10730 (50 pmol) 0.5 μL Probe 10731 (50pmol)   1 μL 10 mM dNTPs   1 μL Taq DNA Polymerase (Promega, M186A)  40μL water

PCR cycling parameters were 96° C., 1 minute; (94° C. 15 seconds; 60° C.30 seconds; 72° C. 45 seconds)×20; 72° C. 45 seconds. The PCR reactionwas purified with 500 μL Wizard™ PCR Purification Resin (Promega, A7181)according to manufacturer instructions and eluted with 25 μL water.

The duplicate DNA targets (1 μL) were then interrogated in duplicate,with 1 μg (200 pmol) probe 10732 (SEQ ID NO:168), a sequence common toboth W3110 and DH5α; 1 μg (200 pmol) probe 10733 (SEQ ID NO:169), asequence completely matching only W3110 DNA; and 1 μg (200 pmol) probe10734 (SEQ ID NO:170), a sequence completely matching only DH5α DNA.Four microliters of the interrogation reaction were combined with 100 μlL/L reagent (Promega, F202A) and the light output measured. hetero-homozygotes zygotes LOH oligo W1 W2 D1 D2 W D W1 W2 D1 D2 alone No oligo93 131 59 115 129 83 101 59 63 71 — 10732 91 372 542 480 447 403 307 53362 352 4 10732 95 465 536 494 479 419 295 257 364 349 4 10733 95 373112 95 251 191 218 173 67 76 6 10733 86 353 108 88 245 187 204 158 77 686 10734 185 212 427 384 337 264 181 161 182 263 111 10734 179 199 409378 318 258 159 127 182 282 111

The deletion-specific interrogation oligonucleotide (10734) gave highbackground. In general these data show the utility of the technology fordetermination of LOH. However, two samples, the first W/W homozygote andfirst D LOH, give aberrant data for an unknown reason. 10730 5′ SEQ IDNO: 166 CACTTTATGCTTCCGGCTCGTATG 3′ (lacZ) 10731 5′GGGATAGGTTACGTTGGTGTAGATGG SEQ ID NO: 167 (lacZ) 10732 5′GTTGGGAAGGGCGATCGGTG 3′ SEQ ID NO: 168 (common lac probe) 10733 5′GGGATGTGCTGCAAGGCGATT 3′ SEQ ID NO: 169 (wt lac probe) 10734 5′GGATTCACTGGCCGTCGTGG 3′ SEQ ID NO: 170 (deletion lac probe)

EXAMPLE 49 Interrogation for Loss of Heterozygosity—CMV

The use of an interrogation assay to determine loss of heterozygositywith a synthetic cytomegalovirus (CMV) target is demonstrated in thisExample.

The CMV target was chosen because the interrogating probeoligonucleotides (9211 (SEQ ID NO:86) and 9212 (SEQ ID NO:35) had beenpreviously used and well characterized. Oligonucleotides 10800 (SEQ IDNO:171) and 10801 (SEQ ID NO:172) were annealed to produce a synthetictarget, “A”, representing a fragment of the CMV genome. Likewise,oligonucleotides 10803 (SEQ ID NO:173) and 10805 (SEQ ID NO:174) wereannealed to produce a synthetic target, “G” representing a fragment ofthe CMV genome. Targets A and G are identical except at one nucleotideposition where they have the nucleotide resulting in their name. Bothtargets have SacI overhangs.

The targets were cloned into the SacI restriction site of pZERO-2plasmid (Invitrogen) and transformed into TOP10 E. coli cells(Invitrogen). The presence of the correct nucleotide sequence in the Aand G clones was confirmed by sequencing. However, the G clone was foundto contain an unintended mutation at the nucleotide position three basesin from the 5′ end of the region that anneals to the interrogationprobes. Because this mismatch is near the 5′ end of the interrogationprobe annealing sequence, it should not affect the interrogationresults.

The following five target solutions were created with the A and Gclones:

-   1. Hetero: 125 pg A and 125 pg G/microliter-   2. LOH A: 125 pg A and no G/microliter-   3. LOH G: no A and 125 pg G/microliter-   4. Mix Ag: 125 pg A and 62 pg G/microliter-   5. Mix Ga: 62 pg A and 125 pg G/microliter

These target solutions were PCR amplified with the JH67 (SEQ ID NO:175)and 11077 (SEQ ID NO: 176) probes in the following reaction:

-   2 μL Target solution-   1 μL Probes JH67 and 11077 (50 pmol each)-   1 μL 10 mM dNTPs-   5 μL 1OX Taq buffer-   1 μL Taq DNA polymerase-   40 μL water

The PCR cycling parameters were: 96° C., 1 minute; (94° C., 15 seconds;60° C., 30 seconds; 72° C. 45 seconds)×15; 72° C. 45 seconds. The entirePCR reaction was then purified with 500 μL Wizard™ PCR purificationresin (Promega, A7170) according to manufacturer's instructions. The DNAwas eluted with 30 μL TE buffer. A standard interrogation reaction with6 μL target and 1 μg interrogation probe, was performed with theexception that 2 units of Klenow exo− were used per reaction. Fourmicroliters of the final reaction were combined with 100 μL of L/Lreagent and the relative light units measured. Oligo Heterozygote LOH ALOH G Mix Ag Mix Ga Alone No oligo 30 40 65 29 34 51 19 59 26 41 — Aoligo 279 340 74 329 27 27 258 309 50 164 5.2 308 372 76 339 20 26 351330 83 167 5.2 G oligo 302 324 37 91 285 272 127 106 245 302 6.3 278 32530 87 256 187 113 124 215 357 6.3 A:G 1.01 1.10 2.26 3.76 0.09 0.11 2.542.78 0.29 0.50 ratio G:A 0.99 0.91 0.44 0.27 11.59 8.71 0.39 0.36 3.461.99 ratio

These data illustrate that LOH can be determined using this method withappropriate interrogation probes. SEQ ID NO: 171 10800 5′CGTGTATGCCACTTTGATATTACACCCATGAACGTG CTCATCGACGTGAACCCGCACAACGAGCT 3′SEQ ID NO: 172 10801 5′ CGTTGTGCGGGTTCACGTCGATGAGCACGTTCATGGGTGTAATATCAAAGTGGCATACACGAGCT 3′ SEQ ID NO: 173 10803 5′CGTGTATGCCACTTTGATATTACACCCGTGAACGTG CTCATCGACGTGAACCCGCCAAACGAGCT 3′SEQ ID NO: 174 10805 5′ CGTTGTGCGGGTTCACGTCGATGAGCACGTTCACGGGTGTAATATCAAAGTGGCATACACGAGCT 3′ SEQ ID NO: 175 JH67 5′TCACACAGGAAACAGCTATGACCATG 3′ SEQ ID NO: 176 11077 5′GCAAGGCGATTAAGTTGGGTAACG 3′ (M13 forward probe) SEQ ID NO: 86 9211 5′CACTTTGATATTACACCCATG 3′ SEQ ID NO: 35 9212 5′ CACTTTGATATTACACCCGTG 3′

EXAMPLE 50 Self-Annealing Interrogation Probe

This Example illustrates use of a different type of oligonucleotideprobe that is used to form a hairpin structure in the interrogationtechnology of this invention. This study demonstrates a method foreliminating the need for adding a probe specific to the interrogationsite to the interrogation reaction.

Here, the oligonucleotide probe anneals to the target strand downstreamof (3′ to) the interrogation position in the target strand. Theoligonucleotide has at its 5′ end an unannealed region of nucleotidesfollowed by about 5 to about 20 nucleotides that are identical to theinterrogation region on the target strand. The annealed 3′ end of theoligonucleotide is then extended through the interrogation position ofthe target strand creating what is referred to as extended probe. Thehybrid is denatured and a hairpin structure formed between the extendedprobe strand and the 5′ end of the oligonucleotide probe. This region isthen assayed in a standard interrogation reaction to determine if amismatch is present or not.

Four probes were designed to represent different types of hairpinformations that an extended probe strands may assume. These probes are10207 (SEQ ID NO:177), 10208 (SEQ ID NO:178), 10209 (SEQ ID NO:179), and10212 (SEQ ID NO:180).

These probes are predicted to form the following self-hybridizedsecondary structures when allowed to self-anneal: 10207 5′A-T-G-A-A-C-G-T-A-C-G-T-C-G-G 3′ T-A-C-T-T-G-C-A             |                   C-C-G-A-G-T-A 10208 5′ G   T-G-A-A-C-G-T-A-C-G-T-C-G-G    A-C-T-T-G-C-A             | 3′T             C-C-G-A-G-T-A 10209        A 5′A-T   A-A-C-G-T-A-C-G-T-C-G-G 3′ T-A   T-T-G-C-A             |       C           C-C-G-A-G-T-A 10212 5′ A-T-A-A-A-C-G-T-A-C-G-T-C-G-G          3′ G-C-A             |                    C-G-A-G-T-A

A 5 μL (5 μg) aliquot of each of the four probes was diluted to 100 μLwith nanopure water. They were then sequentially diluted 1:10 to a finaldilution factor of 1:100,000. Twenty microliters of the diluted probeswere heated, in separate tubes, at 95° C. for 3 minutes and cooled toroom temperature for 10 minutes to permit self-annealing. Twentymicroliters of Master Mix, as described in Example 1, were then added toeach tube and the tubes were incubated at 37° C. for 15 minutes. Tenmicroliters of the solutions were added to 100 μL of L/L reagent(Promega, F202A) and relative light units measured immediately with aTurner® TD20/20 luminometer. The no-probe control resulted in 57.24relative light units and the remaining probe results are reported belowin relative light units (rlu). Log Probe dilution 10207 10208 1020910212 −5 44.89 56.22 57.57 57.80 −4 85.21 64.56 58.26 63.15 −3 297.770.53 79.12 82.65 −2 970.5 108.4 80.06 106.7

Probe 10207 worked as an efficient target for interrogation as expected,with probe 10208 providing the anticipated negative results. Probe 10212has only a three base match so it may be un-extended, thus resulting inthe low values. Probe 10209 likely has the 3′ terminal nucleotideunannealed when the hairpin forms due to the mismatch at the thirdnucleotide in from the 3′ end. Such an unannealed 3′ terminal nucleotidewould account for the low rlu values. 10207 5′ATGAACGTACGTCGGATGAGCACGTTCAT 3′ SEQ ID NO: 177 10208 5′GTGAACGTACGTCGGATGAGCACGTTCAT 3′ SEQ ID NO: 178 10209 5′ATAAACGTACGTCGGATGAGCACGTTCAT 3′ SEQ ID NO: 179 10212 5′ATAAACGTACGTCGGATGAGCACG 3′ SEQ ID NO: 180

EXAMPLE 51 Determination of Viral Load

This Example illustrates that the presence of viral nucleic acid inserum samples can be determined to a detection level of ten copies ofviral nucleic acid per sample.

Hepatitis C Virus (HCV) RNA was isolated from infected or uninfectedhuman serum samples. A two-step RT-PCR was performed using HCV-specificprobes and about 1000 viral equivalents of RNA, and samples wereinterrogated using the interrogation probe HCV1 (SEQ ID NO:181).

Two HCV positive samples, one HCV negative sample, and a water controlwere analyzed in duplicate. The interrogation reaction was added to 100μL of L/L reagent (Promega F202A) and the light output measuredimmediately on a Turner® TD20/20 luminometer. The average relative lightunit values were as follows. Water control 38.6 HCV minus 239.0 HCVpositive (1) 1261.0 HCV positive (2) 1390.0

To determine the sensitivity of viral detection using this technology,RT-PCR was performed on HCV positive and HCV negative controls as wellas samples estimated to contain 1000, 100, and 10 viral RNA copies.Twenty five microliters of each amplification reaction were purifiedusing magnetic silica as described in Example 38 and eluted in 100 μLwater. Four microliters of the eluted DNA were interrogated using theinterrogation probe described above in a standard interrogation reactionas described in Example 32. The interrogation reaction was added to 100μL of L/L reagent and the light output measured on a Turner® TD 20/20luminometer. Ten copies of HCV are readily detected in this assay. Theaverage relative light unit (rlu) values were as follows. Sample rluWater 49.0 Water 54.2 HCV neg control 59.4 HCV neg control 62.1 HCV poscontrol 653.7 HCV pos control 743.1 HCV 1000 copies 460.7 HCV 1000copies 429.5 HCV 100 copies 405.1 HCV 100 copies 404.3 HCV 10 copies184.9 HCV 10 copies 179.5

HCV1: 5′ CTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGG 3′ SEQ ID NO: 181

EXAMPLE 52 Interrogation of DNA Sequences from Genetically ModifiedOrganisms

According to European Union (EU) Regulation on Novel Foods and NovelFood Ingredients, adopted in 1997, genetically modified foods must belabeled as such if they are “no longer equivalent” to their conventionalcounterparts. This includes when the foods have a different composition,use or nutritional value from the conventional food. The EU subsequentlydecided that the presence of just a fragment of genetically modifiedprotein or DNA is enough to make the product “no longer equivalent” toconventional products for soya and maize and, therefore, such productsrequire labeling.

Genetically modified organisms (GMO), particularly plants, are oftengenetically modified to include the exogenous specific DNA of interestalong with an exogenous transcription sequence such as the 35S promoterand the NOS terminator. In this example, the DNA of soya and maizesamples are analyzed for the presence or absence of the 35S promoter andNOS terminator. The PCR Primers 35S-1 (SEQ ID NO:182) and 35S-2 (SEQ IDNO:183) were used to prepare a 235 bp PCR product. The Primers NOS-1(SEQ ID NO:184) and NOS-2 (SEQ ID NO:185) were used to prepare a 220 bpPCR product.

GMO positive and negative control DNA (20 ng) were PCR amplified using50 pmol of the 35S promoter and NOS terminator PCR primer pairs. The PCRcycling profile was 94° C., 3 minutes; (94° C., 30 seconds; 54° C., 40seconds; 72° C., 1 minute)×40; 72° C., 3 minutes. The resulting PCRproducts (25 μL) were purified using magnetic silica and eluted in 100μL water as described in Example 33. Four microliters of the eluted PCRproducts were used in a standard interrogation assay as described inExample 32 and the relative light unit (rlu) results are detailed in thefollowing table. The 35S interrogation probes used were 11211 (SEQ IDNO:186) and 11210 (SEQ ID NO:187). The NOS interrogation probes usedwere 11212 (SEQ ID NO:188) and 11213 (SEQ ID NO:189). PCR InterrogationDNA Oligos Oligos rlu GMO minus, soy 35S 11210 166.6 GMO minus, soy 35S11210 172.0 GMO minus, soy 35S 11211 206.8 GMO minus, soy 35S 11211205.8 GMO minus, soy 35S none 95.7 GMO minus, maize 35S 11210 245.0 GMOminus, maize 35S 11210 254.3 GMO minus, maize 35S 11211 271.3 GMO minus,maize 35S 11211 275.7 GMO minus, maize 35S none 116.0 GMO positive, soy35S 11210 1456.0 GMO positive, soy 35S 11210 1442.0 GMO positive, soy35S 11211 1546.0 GMO positive, soy 35S 11211 1529.0 GMO positive, soy35S none 865.0 GMO positive, maize 35S 11210 1252.0 GMO positive, maize35S 11210 1299.0 GMO positive, maize 35S 11211 1358.0 GMO positive,maize 35S 11211 1361.0 GMO positive, maize 35S none 705.6 GMO minus, soyNOS 11212 73.9 GMO minus, soy NOS 11213 75.8 GMO minus, soy NOS none76.1 GMO positive, soy NOS 11212 615.0 GMO positive, soy NOS 11213 616.6GMO positive, soy NOS none 98.0

The above data demonstrate that the interrogation reaction works for theidentification of presence or absence of GMO DNA in DNA samples isolatedfrom soy and maize products. The 35S PCR product gave high backgroundvalues by itself, which can be reduced by using a primer withphosphorothioate linkages near the 5′-terminus for the PCR reactionfollowed by exo6 treatment to remove one strand of the PCR product asdescribed in Example 38 and below. The PCR primers 35S1 and NOS1 wereresynthesized to have phosphorothioate linkages between the first fivebases at the 5′ end. The PCR reaction was repeated and the resulting PCRproduct treated with exo6 and purified as described in Example 38.

Four microliters of the purified DNA were used for the standardinterrogation assay using the NOS primer 11212 and the 35S primer 11211with 5 units of Klenow exo−. The rlu data obtained are in the tablebelow. PCR Interrogation DNA oligos oligo rlu GMO minus, soy NOS 1121252.3 GMO minus, soy NOS 11211 60.2 GMO minus, soy NOS none 53.3 GMOpositive, soy NOS 11212 277.1 GMO positive, soy NOS 11211 84.4 GMOpositive, soy NOS none 75.7 GMO minus, soy 35S 11212 57.8 GMO minus, soy35S 11211 66.9 GMO minus, soy 35S none 54.6 GMO positive, soy 35S 1121299.7 GMO positive, soy 35S 11211 397.6 GMO positive, soy 35S none 86.0GMO positive, soy 35S + NOS 11212 249.4 GMO positive, soy 35S + NOS11211 290.1 GMO positive, soy 35S + NOS 11211 + 11212 482.5 GMOpositive, soy 35S + NOS none 70.5

This method greatly reduced the background from the 35S PCR product andpermitted better discrimination between the GMO positive and GMO minusDNA samples. Also, this example again demonstrates the utility of thetechnology for multiplexing both the PCR reaction and the interrogationreaction. As seen in the last four reactions above, the data show thatthe use of multiple PCR probes and/or multiple interrogation probesleads to identification of GMO organisms.

35S promoter PCR primers: 35S-1 5′ GATAGTGGGATTGTGCGTCA 3′ SEQ ID NO:182 35S-2 5′ GCTCCTACAAATGCCATCA 3′ SEQ ID NO: 183 NOS terminator PCRprimers NOS-1 5′ TTATCCTAGTTTGCGCGCTA 3′ SEQ ID NO: 184 NOS-2 5′GAATCCTGCTGCCGGTCTTG 3′ SEQ ID NO: 185

35S Interrogation oligonucleotide probes: 11211 5′ GCAAGTGGATTGATG 3′SEQ ID NO: 186 11210 5′ CCAACCACGTCTTCAAA 3′ SEQ ID NO: 187 NOSInterrogation oligonucleotide probes 11212 5′ TTTATGAGATGGGTTT 3′ SEQ IDNO: 188 11213 5′ ATGATTAGAGTCCCG 3′ SEQ ID NO: 189

EXAMPLE 53 Pyrophosphorylation Chain Reaction: High SensitivityDetection Through Generation of New Targets by Probe Design

Pyrophosphorylation of a DNA/DNA or a DNA/RNA hybrid with MMLV RT andKlenow Exo− does not take place when the duplex has a 3′ overhang. Twoprobes designed to hybridize to the target exactly, but also to eachother to yield 3′ overhangs, do not increase the background when presentin the pyrophosphorylation reaction because they are not substrates forthese enzymes when hybridized to each other.

If the hybridized, treated sample is subjected to thepyrophosphorylation reaction, the ends of the probe on the targetsequence are pyrophosphorylated. In this example, the presumption hasbeen made that 5 bp are removed from these probes. After the reaction iscomplete, a total of “two ends worth” of nucleoside triphosphate isformed.

Heating the reaction components to 95° C. denatures the hybrids from thefirst round. When the reaction is cooled, the denatured hybrid strandsbecome available for hybridization to other probes. This reaction is nowsubjected to a second round of pyrophosphorylation. The probeshybridized to the target sequence again pyrophosphorylate generatinganother “two ends worth” of nucleoside triphosphate. In addition, theoriginal probes that have hybridized to unpyrophosphorylated probes aresubstrates for the reaction and also contribute “two ends worth” ofnucleoside triphosphate. Thus, a total of “four ends worth” ofnucleoside triphosphate are formed in this round. This is in addition tothe “two ends worth” from round one, so a total of six ends worth arepresent after round two.

Cycling can continue for additional rounds. At the end of round 3 atotal of 12 ends worth of nucleoside triphosphate are formed. At the endof round 4, a total of 20 ends worth of nucleotide triphosphate areformed and at the end of round 5, a total of 30 ends worth of nucleotidetriphosphate are formed.

EXAMPLE 54 Pyrophosphorylation Chain Reaction II

The RNA concentration limit for detection is considerably higher thanDNA, which can be routinely detected in lower concentrations than RNA.This study demonstrates a way to increase nucleic acid detectionsensitivity using a pyrophosphorylation chain reaction.

Here, a low signal level from the initial reaction is augmented using aseries of probes that can be substrates for pyrophosphorylation reactiononly if the 3′ end of the target is resultant from a previouspyrophosphorylation reaction. Unless there is pyrophosphorylation of theprevious oligonucleotide, it would have a 3′ overhang when hybridized tothe second round oligonucleotide and thus would not function as asubstrate for the pyrophosphorylation reaction with Klenow exo− enzymeor MMLV RT.

Two probes, 9225 (SEQ ID NO:190) and 9276 (SEQ ID NO:191), wereprepared, each of which is complementary to probe 8272 (SEQ ID NO:192)and each of which is recessed on the 5′ end when hybridized with probe8272. Probe 9225 is recessed 2 bases and probe 9276 is recessed 1 base.When probe 8272 hybridizes with either 9225 or 9276, it is not asubstrate for Klenow exo− or MMLV RT because there is a 3′ overhang.However, if probe 8272 is previously pyrophosphorylated (e.g. whenhybridized to mRNA), it will create 5′ overhangs when hybridized to 9225or 9276. Therefore, the second cycle of pyrophosphorylation enhancespyrophosphorylation values. Probe 8271 (SEQ ID NO:193) is used as apositive control, as it is an exact complement of probe 8272 and it is apyrophosphorylation substrate for Klenow Exo− regardless of whether ornot probe 8272 is previously pyrophosphorylated.

The following enzyme mix was assembled:  36 μL 5 × MMLV-RT buffer(Promega, M531A) 4.5 μL 40 mM NaPPi (Promega, C113A) 9.0 μL 200 U/μLMMLV-RT (Promega, M1701)

First cycle pyrophosphorylation reactions were set up to include thefollowing. Reaction A Reaction B Reaction C  10 mM Tris, pH 7.3 41.5 μL42.5 μL 40.5 μL  50 ng/μL Globin mRNA   2 μL none   2 μL (Gibco BRL cat#18103-028) 100 ng/μL probe 8272 none   1 μL   1 μL enzyme mix 16.5 μL16.5 μL 16.5 μL

The three reactions were incubated at 37° C. for 60 minutes then 15 μLof each reaction were aliquoted into 4 separate tubes. For each set ofreactions, 2 μL Tris, 2 μL probe 9225, 2 μL probe 9276, or 2 μL probe8271 were added and the tubes were then further incubated at 55° C. for15 minutes and cooled to room temperature. This is referred to as thesecond cycle hybridization mix.

A master mix for the second round of pyrophosphorylation was assembledas follows: 426 μL water  80 μL 10 × Pol Buffer  10 μL 40 mM NaPPi  4 μLNDPK (1 U/μL)  40 μL 2 mM ADP (Sigma)  40 μL Klenow Exo−

Fifteen microliters of master mix were added to 5 μL of each secondcycle hybridization mix. These tubes were then incubated at 37° C. for30 minutes. Thereafter, 100 μof L/L reagent (Promega F202A) were addedand the relative light units were measured on a Turner® TD20/20luminometer.

The average of triplicate measurements is reported below in relativelight units (avg. rlu). 1^(st) cycle 2^(nd) cycle probe avg. rlu mRNAonly none 22.16 mRNA only 9225 54.82 mRNA only 9276 67.28 mRNA only 8271198.00 Probe 8272 only none 42.74 Probe 8272 only 9225 186.13 Probe 8272only 9276 910.13 Probe 8272 only 9271 1252.67 mRNA + 8272 none 127.97mRNA + 8272 9225 641.17 mRNA + 8272 9276 1152.67 mRNA + 8272 92711122.33

Thus, after the removal of the 3′ overhang due to pyrophosphorylation,probe 8272 hybridized with secondary probes becoming a substrate for2^(nd) round of pyrophosphorylation. However, the results are confuseddue to an unknown reason by high background. Even without MRNA present,unpyrophosphorylated probe 8272, when combined with either probe 9225 orprobe 9276 provided elevated 2^(nd) cycle rlu values. 9225 5′GGTGCATCTGTCCAGTGAGGAGAAGTCTGC 3′ SEQ ID NO: 190 9276 5′TGGTGCATCTGTCCAGTGAGGAGAAGTCTG 3′ SEQ ID NO: 191 8272 5′AGACTTCTCCTCACTGGACAGATGCACCAT 3′ SEQ ID NO: 192 8271 5′ATGGTGCATCTGTCCAGTGAGGAGAAGTCT 3′ SEQ ID NO: 193

EXAMPLE 55 Dual Probe Rolling Circle Amplification Prior toInterrogation

The amplification of target nucleic acid by means of rolling circleamplification prior to the interrogation reaction is examined in thisExample as a substitute methodology for PCR amplification. A typicalrolling circle amplification of a circular target using two probes isdescribed in Lizardi, P. M. et al. Nature Genetics, 19:227 (1998).

The wild type target used in this study is oligonucleotide 10870 (SEQ IDNO:194). The mutant target used is oligonucleotide 10994 (SEQ IDNO:195). The open circle probe which anneals to the targets isoligonucleotide 10865 (SEQ ID NO:196). Rolling circle replication primerwhich anneals to the open circle probe is oligonucleotide 10866 (SEQ IDNO:197). Rolling circle replication primer 10869 (SEQ ID NO:198) has a3′-terminal residue that anneals only to the wild type target, whereasprobe 10989 (SEQ ID NO:199) has a 3′-terminal residue that anneals onlyto the mutant target. Probes 10869 and 10989 both have a nucleotidelocated 3 nucleotides from the 3′-terminal nucleotide that is notcomplementary to either the wild type or the mutant target. Thismismatched base was intentionally incorporated to provide for increasedspecificity in the interrogation reaction.

This Example uses a synthetic heterozygote solution containing both the10870 and 10994 oligonucleotides as targets. The 10865 oligonucleotideanneals to both of these targets in a similar manner, forming a circulartarget with a seven base pair gap that needs to be filled in from the10865 3′ end and ligated in order for complete rolling circleamplification to proceed. The annealing of the 10865 oligonucleotide to10870 wild type and 10994 mutant oligonucleotides is diagrammed inFIG. 1. In the absence of ligation, no priming can occur.

The following heterozygote solution was assembled:   2 μL 10 × ampligasebuffer (Epicenter)   2 μL 2 mM dTTP, dCTP, dATP   1 μL 500 μg/mL probe10865 (probe) 0.5 μL 500 μg/mL probe 10870 (wild type target strand) 0.5μL 500 μg/mL probe 10994 (mutant target strand)   1 μL 5 u/μL Tfl DNApolymerase   1 μL 5 u/μL Ampligase (Thermostable ligase, Epicenter)  12μL water

Likewise, the homozygote solutions were assembled using either 1 μLprobe 10870 and no 10994 to prepare the wild type homozygote target, orno 10870 and 1 μL 10994 to prepare the mutant homozygote target. Onlythree deoxynucleotides are included in the solutions to prevent stranddisplacement. The solutions were incubated at 65° C. for 30 minutesafter which time they were estimated to have formed about 2.5×10¹¹circular molecules per microliter. After the 30 minutes of incubationthey were diluted to about 2500 circular targets per microliter. Theheterozygote, the homozygote wild type and homozygote mutant gap-fillligations were then used to assemble the following amplificationreactions. Reaction: 1 2 3 4 5 6   1 μL WT* WT Mut* Mut Target homoZ*homoZ homoZ homoZ heteroZ* heteroZ 0.5 μL Probe 10869 10989 10869 1098910869 10989 WT Mut WT Mut WT Mut*WT = wild type; Mut = mutant; homoZ = homozygote; and heteroZ =heterozygote

Additionally, each tube contained 2 μL 10× Polymerase buffer, 1 μL 10 mMdNTPs, 0.5 μL probe 10866, 1 μL 1.2 μg/μL T4 Gene 32, 1.5 μL DMSO and18.5 μL water. The assembled components were then put on ice and 3 μLVent exo− (2 U/μL) were added. The solutions were covered with 30 μL ofmineral oil and placed at 95° C. for 3 minutes then 65° C. for 90minutes. The free nucleotides were then removed using the Wizard™ PCRpurification system (Promega, A7170) and the DNA eluted with 50 μLwater.

The following Master mix was assembled.   30 μL 10 × Buffer A 3.75 μL 40mM NaPPi   15 μL T4 DNA polymerase (10 U/μL)   3 μL NDPK (1 U/μL)   3 μL10 μM ADP (Sigma)  245 μL water

One microliter of the above amplification reactions was added to 19 μLmaster mix in duplicate tubes. The tubes were incubated for 15 minutesat 37° C., then 5 μL of the reaction were added to 100 μL of L/L reagent(Promega F202A) and light output was measured in a Turner® TD20/20luminometer. Relative light units Undiluted 1:4 diluted Second/ Rxn*target target probe Target 1. 618.6 458.2 WT* WT homoZ* 1. 621.6 457.7WT WT homoZ 2. 282.4 90.2 Mut* WT homoZ 2. 288.4 100.3 Mut WT homoZ 3.365.9 148.6 WT Mut homoZ 3. 379.8 149.9 WT Mut homoZ 4. 632.5 461.5 MutMut homoZ 4. 650.2 442.4 Mut Mut homoZ 5. 606.1 381.1 WT heteroZ* 5.608.6 394.3 WT heteroZ 6. 631.0 420.1 Mut heteroZ 6. 637.3 411.4 MutheteroZ*Rxn = reaction; WT = wild type; Mut = mutant; homoZ = homozygote; andheteroZ = heterozygote

At the lower amount of target DNA, the mutant:wild type discriminationimproves to 3-5 fold from the 2-3 fold exhibited when using theundiluted target DNA. This indicates that the study using the undilutedtarget was likely out of the linear range. The heterozygote ratio forboth studies is close to the expected 1:1. 10870 5′TTGCAGAGAAAGACAATATAGTTCTTGGAGAAGGTGGAA TCACACTGAGTGGA 3′ SEQ ID NO: 19410994 5′ TTGCAGAGAAAGACAATATAGTTCTTTGAGAAGGTGGAATC ACACTGAGTGGA 3′ SEQID NO: 195 10865 5′ GAACTATATTGTCTTTCTCTGATTCTGACTCGTCATGTCTCAGCTTTAGTTTAATACGACTCACTATAGGGCTCAGTGTGATTCCACCT 3′ SEQ ID NO: 196 108665′ CTAAAGCTGAGACATGACGAGTC 3′ SEQ ID NO: 197 10869 5′CTCAGTGTGATTCCACCTTCACC 3′ SEQ ID NO: 198 10989 5′CTCAGTGTGATTCCACCTTCACA 3′ SEQ ID NO: 199

EXAMPLE 56 Tne Triple Mutant Tne Polymerase and Thermostable NDPK Usedto Interrogate Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessivediseases resulting from a wide range of mutations in the steroid21-hydroxylase (CYP21) gene that contains 10 exons. There is a highlevel of nucleic acid homology (98% in exons, 96% in introns) betweenCYP21, the functional gene, and CYP21P, the nonfunctional pseudogene.The many types of mutations in this gene that can lead to diseaseinclude complete gene deletions, large gene conversions, single pointmutations, and a small 8 bp deletion [See, White, et al., Hum. Mutat.,3:373-378, (1994)].

The majority of the CAH disease-causing mutations are sequences presentin the nonexpressed CYP21P pseudogene, and arise in the CYP21 genethrough recombination between CYP21P and CYP21. Thus, one mutationdetection strategy specifically detects the CYP21 gene, and not theCYP21P pseudogene. The frequency of disease-carrying alleles in thepopulation is about 1 in 50.

In this example, the CAH target was interrogated for a variety ofmutations using Klenow exo− and yeast NDPK, and the results werecompared to a similar analysis using Tne triple mutant thermostable DNApolymerase and a thermostable Pfu NDPK. Both wild type CAH PCR products,mutant synthetic targets, and a pseudogene PCR product amplified fromthe cloned CYP21P pseudogene were utilized as targets in this assay.They are listed below.

Primer pairs used in PCR amplification and the resulting products are asfollows. Size PCR Segment Primers Segment Amplified 10912 + 10909 1400bp 5′ end CYP21 11461 + 11480  918 bp 5′ end CYP21 10910 + 11286 1492 bp3′ end CYP21 11535 + 11286 1496 bp 3′ end CYP21 10912 + 10911 2680 bppseudogene (CYP21P)

Synthetic targets and interrogation oligos utilized are listed below.

PCR reactions were assembled to amplify regions of the CAH gene with 4different probe sets, using undigested human genomic DNA (Promega,G3041) as target (25 ng per reaction). For amplification of thepseudogene, human genomic DNA was predigested with the restrictionenzyme Bcl I, which specifically cleaves the CYP21 gene upstream of theforward PCR probe, thus permitting only amplification of CYP21P [Krone,Clinical Chem. 44(10):2075-2082 (1998)].

The 2680 bp PCR product was amplified from 50 ng of digested DNA andsubsequently cloned into the plasmid vector PGEM-T Easy (Promega, A1380)following the manufacturer's protocol. A clone was selected andsequenced (USB Sequenase kit, US70770) to confirm it was indeed thepseudogene. The cloned CYP21P gene in the pGEM-T Easy vector was used insubsequent amplifications to obtain pure pseudogene PCR product formutation interrogation analysis (100 pg of plasmid per PCR reaction).

All 50 μL amplification reactions contained the following reagents:genomic DNA (as described above), 1× reaction buffer (M1901), 1.0-1.5 mMmagnesium chloride (all with 1.0 mM except probe pair 10912+10911 forpseudogene, which contained 1.5 mM MgCl₂; Promega, A3511), 200 μM eachdNTP (C1141), 50 pmoles each probe, and 2.5 units Taq DNA Polymerase(M1665).

The following cycling profile was utilized for all amplifications: 5minutes at 95° C.; 40 cycles of 30 seconds at 94° C., 1 minute at 55°C., 1 minute per kbp of product at 72° C.; 8 minutes at 68° C.; soak at40° C. The products were analyzed on 1% agarose gels and compared to DNAmolecular weight standards to confirm product sizes were correct. Analiquot of each PCR reaction (25 μL) was then treated with 50 units T7Gene6 Exonuclease (USB, E70025Y) for 15 minutes at 37° C., followed bypurification using the Wizard™ PCR Prep DNA Purification System(Promega, A7170) with 3×1 mL 80% isopropanol washes. Theexonuclease-treated DNA was eluted in 100 μL of nuclease-free water.

Each interrogation assay (20 μL total volume) contained 4 μL of purifiedPCR product or 5 ng of synthetic target, and 1 μg interrogation oligoprobe (or water for the no-oligo background control). The reactions wereincubated at 95° C. for 3 minutes, followed by 10 minutes at 37° C. forKlenow exo− or 55° C. for Tne polymerase. Twenty microliters of mastermix were added (2 mM sodium pyrophosphate, 0.2 μM ADP, 2× polymerasebuffer (M195A for Klenow or M1901 for Tne), 5 mM magnesium chloride forTne only, 1-2 U Klenow exo− and 0.2 U yeast NDPK or 1 U Tne triplemutant polymerase and 0.1 U Pfu NDPK) and the reaction incubated 15minutes at 37° C. (Klenow exo−) or 55° C. (Tne). The entire reaction wasthen added to 100 μL of L/L reagent (Promega FF202A) and light outputread in a Turner® TD20/20 luminometer.

Although 55° C. was used in these studies with the Tne triple mutantpolymerase and the Pfu NDPK, higher temperatures can also be used. The55° C. temperature selected appeared to be a good compromise betweeninterrogation oligo annealing and enzymatic activity. Thus, higherincubation temperatures can be beneficial if longer interrogation oligosare utilized.

The table below contains the relative light units (rlu) obtained. Thedata represent the combined results of many separate studies using thevarious enzymes. The use of the Tne triple mutant polymerase and PfuNDPK particularly improved the discrimination ratio for the CAH wildtype PCR products at mutation sites 2 and 6, whereas the thermostableenzymes improved the discrimination ratio for the mutant pseudogene PCRproduct at mutation sites 3, 4, and 5. The synthetic targets worked wellwith both enzymes, however the signals and discrimination ratios werehigher for the thermostable enzymes at almost all of the mutation sites.Tne/ Tne/ Tne/ Klenow/ Klenow/ Klenow/ Pfu Pfu Pfu NDPK NDPK NDPK NDPKNDPK NDPK Target No WT Mutant No WT* Mutant Mut* DNA oligo oligo oligooligo oligo oligo Site CAH WT 176.9 1050.0 204.0 1 1400 bp CAH WT 176.91149 625.5 2 1400 bp CAH WT 388.9 496.2 414.7 3 1492 bp CAH WT 388.9881.4 383.9 4 1492 bp CAH WT 388.9 940.3 477.3 5 1492 bp CAH WT 388.9205.2 207.0 6 1492 bp CAH WT 129.4 443.0 125.6 1 918 bp CAH WT 129.4440.9 134.7 2 918 bp CAH WT 124.3 261.6 118.3 3 1496 bp CAH WT 124.3259.4 121.7 4 1496 bp CAH WT 124.3 276.3 135.6 5 1496 bp CAH WT 124.3214.0 112.3 6 1496 bp CAH WT 124.3 252.5 174.5 7 1496 bp Pseudogene15.89 115.7 109.2 176.1 419.0 537.3 1 2680 bp Pseudogene 15.89 45.29140.4 176.1 388.6 397.5 2 2680 bp Pseudogene 15.89 129.6 141.6 176.1477.8 772.5 3 2680 bp Pseudogene 15.89 63.34 149.2 176.1 369.4 999.7 42680 bp Pseudogene 15.89 115.8 91.28 1676.1 412.1 945.9 5 2680 bpPseudogene 176.1 202.7 945.0 7 2680 bp Synthetic 95.65 128.2 831.2 56.9276.58 1499 1 Temp. 1* Synthetic 81.09 119.3 774.9 58.46 171.8 1521 2Temp. 2 Synthetic 83.22 315.6 1496 54.05 171.2 2206 3 Temp. 3 Synthetic87.71 85.82 1199 55.29 152.1 2829 4 Temp. 4 Synthetic 78.80 332.5 107157.49 76.91 837.7 5 Temp. 5 Synthetic 79.86 57.0 322.0 56.68 140.6 23286 Temp. 6 Synthetic 86.99 1738 1285 209.2 4162 351.3 2 Temp. 7 Synthetic98.50 1005 29.24 212.2 2121 260.4 6 Temp. 8*WT = wild type; Mut = mutation; Temp. = template

PCR Primers Utilized: 10909 5′ CCAGAGCAGGGAGTAGTCTC 3′ SEQ ID NO: 200CAH reverse primer; 5′ most 3 linkages phosphorothioate (CYP21 only)10912 5′ GCATATAGAGCATGGCTGTG 3′ SEQ ID NO: 201 CAH forward primer 109105′ CCTGTCCTTGGGAGACTAC 3′ SEQ ID NO: 202 CAH forward primer (CYP21 only)10911 5′ CCCAGTTCGTGGTCTAGC 3′ SEQ ID NO: 203 CAH reverse primer; 5′most 3 linkages phosphorothioate 11286 5′ TCCTCACTCATCCCCAAC 3′ SEQ IDNO: 204 CAH reverse primer; 5′ most 3 linkages phosphorothioate 11461 5′GAAATACGGACGTCCCAAGGC SEQ ID NO: 205 CAH forward primer 11480 5′CTTTCCAGAGCAGGGAGTAG SEQ ID NO: 206 CAH reverse primer; 5′ most 3linkages phosphorothioate (CYP21 only) 11535 5′ CCGGACCTGTCCTTGGGAGA SEQID NO: 207 CAH forward primer (CYP21 only)

Synthetic Targets Utilized: 11293 5′AGAAGCCCGGGGCAAGAGGCAGGAGGTGGAGGCTCCGGAG 3′ SEQ ID NO: 208 CAH SyntheticTarget 1 for Interrogator oligo 1 (pseudogene/mutant - exon 1) Mutationsite 1 11294 5′ AGCTTGTCTGCAGGAGGAGCTGGGGGCTGGAGGGTGGGAA 3′ SEQ ID NO:209 CAH Synthetic Target 2 for Interrogator oligo 2 (pseudogene/mutant -intron 2) Mutation site 2 11295 5′TCCGAAGGTGAGGTAACAGTTGATGCTGCAGGTGAGGAGA 3′ SEQ ID NO: 210 CAH SyntheticTarget 3 for Interrogator oligo 3 (pseudogene/mutant - exon 4) Mutationsite 3 11296 5′ TCCACTGCAGCCATGTGCAAGTGCCCTTCCAGGAGCTGTC 3′ SEQ ID NO:211 CAH Synthetic Target 4 for Interrogator oligo 4 (pseudogene/mutant -exon 7) Mutation site 4 11297 5′TCGTGGTCTAGCTCCTCCTACAGTCGCTGCTGAATCTGGG 3′ SEQ ID NO: 212 CAH SyntheticTarget 5 for Interrogator oligo 5 (pseudogene/mutant - exon 8) Mutationsite 5 11298 5′ GCTAAGGGCACAACGGGCCACAGGCGCAGCACCTCGGCGA 3′ SEQ ID NO:213 CAH Synthetic Target 6 for Interrogator oligo 12(pseudogene/mutant - exon 8) Mutation site 6 11484 5′CAGCTTGTCTGCAGGAGGAGTTGGGGGCTGGAGGGTGGGA 3′ SEQ ID NO: 214 CAH SyntheticTarget 7 for Interrogator oligo 7 (wild type - intron 2) Mutation site 211485 5′ GGCTAAGGGCACAACGGGCCGCAGGCGCAGCACCTCGGCG 3′ SEQ ID NO: 215 CAHSynthetic Target 8 for Interrogator oligo 11 (wild type - exon 8)Mutation site 6

Interrogation Oligos Probes Utilized: 11143 5′ CGGAGCCTCCACCTCCCG 3′ SEQID NO: 216 CAH interrogator oligo 6 (wild type) for mutation site 111085 5′ CACCCTCCAGCCCCCAGC 3′ SEQ ID NO: 217 CAH interrogator oligo 2(pseudogene/mutant) for mutation site 2 11084 5′ CGGAGCCTCCACCTCCTG 3′SEQ ID NO: 218 CAH interrogator oligo 1 (pseudogene/mutant) for mutationsite 1 11086 5′ CCTCACCTGCAGCATCAAC 3′ SEQ ID NO: 219 CAH interrogatoroligo 3 (pseudogene/mutant) for mutation site 3 11144 5′CACCCTCCAGCCCCCAAC 3′ SEQ ID NO: 220 CAH interrogator oligo 7 (wildtype) for mutation site 2 11145 5′ CCTCACCTGCAGCATCATC 3′ SEQ ID NO: 221CAH interrogator oligo 8 (wild type) for mutation site 3 11087 5′CCTGGAAGGGCACTT 3′ SEQ ID NO: 222 CAH interrogator oligo 4(pseudogene/mutant) for mutation site 4 11146 5′ CCTGGAAGGGCACGT 3′ SEQID NO: 223 CAH interrogator oligo 9 (wild type) for mutation site 411088 5′ GATTCAGCAGCGACTGTA 3′ SEQ ID NO: 224 CAH interrogator oligo 5(pseudogene/mutant) for mutation site 5 11147 5′ GATTCAGCAGCGACTGCA 3′SEQ ID NO: 225 CAH interrogator oligo 10 (wild type) for mutation site 511287 5′ CGAGGTGCTGCGCCTGCG 3′ SEQ ID NO: 226 CAH interrogation oligo 11(wild type) for mutation site 6 11288 5′ CGAGGTGCTGCGCCTGTG 3′ SEQ IDNO: 227 CAH interrogation oligo 12 (pseudogene/mutant) for mutation site6 11641 5′ GGGATCACATCGTGGAGATG 3′ SEQ ID NO: 228 CAH interrogationoligo 23 (wild type) for mutation site 7 11642 5′ GGGATCACAACGAGGAGAAG3′ SEQ ID NO: 229 CAH interrogation oligo 24 (pseudogene/mutant) formutation site 7

EXAMPLE 57 Multiplex Analysis of Congenital Adrenal Hyperplasia (CAH)Gene

The use of thermostable enzymes to interrogate the CAH gene, asdescribed in Example 56, has also permitted the interrogation of up to 6multiple sites within one reaction. The method used in this Example isillustrative of routine studies carried out in screening laboratorieswhere usual results show the presence of an expected gene (or theabsence of a mutant gene) in almost all of the samples, and only rarelyshows the presence of a mutant gene. In the case illustrated here, aqualitative result is provided from which the exact mutation present canbe determined in a subsequent assay.

Thus, equal volumes of the CAH wild type (WT) 918 bp and 1496 bp PCRproducts (see Example 56) were combined (to thus span the entire CAHgene) and interrogated either separately at each mutation site, or as amultiplexed group. The discrimination ratio was good both in theseparate reactions for the combined PCR products, as well as themultiplexed reaction. In addition, the multiplexed reaction using theCAH wild type PCR products and either 6 wild type interrogation oligoprobes or 6 mutant interrogation oligo probes was combined with anequimolar amount of synthetic target (mutant synthetic target for eachmutation site; 0.2 pmoles either PCR product or synthetic target), tosimulate a heterozygote sample. Tne/Pfu Tne/Pfu Tne/Pfu Probe MutantNDPK, NDPK, NDPK for Synthetic Target No WT Mutant Mutation Target DNAOligo Oligo Oligo Site Added CAH WT 172.7 553.0 180.2 1 918 bp + 1496 bpSame 172.7 535.7 184.0 2 Same 172.7 494.8 182.0 3 Same 172.7 486.7 148.74 Same 172.7 471.7 187.9 5 Same 172.7 317.5 179.7 6 Same 172.7 297.5246.4 7 Same 523.7 1929.0 499.5 1, 2, 3, 4, 5 and 6 Same 506.0 1882.02234.0 1 1 Same 525.4 1848.0 1505.0 2 2 Same 535.9 1735.0 2877.0 3 3Same 547.5 1880.0 4879.0 4 4 Same 552.4 2000.0 3864.0 5 5 Same 482.91938.0 2189.0 6 6 Same 514.5 1791.0 4192.0 2 + 4 2 + 4 Same 537.6 1752.03427.0 5 + 6 5 + 6

Because of the large size of the CAH gene and the large number ofdifferent mutations that may be present, the use of the thermostableenzymes, and thus the increased stringency of the detection procedure,was found to be highly advantageous with this complex target. Mutationsites that interrogated poorly using Klenow exo− and yeast NDPK at 37°C., were more successfully interrogated when using the Tne triple mutantpolymerase and Pfu NDPK at elevated temperatures. In addition, use ofthe thermostable enzymes permitted the multiplexing of numerous wildtype or mutant interrogation oligos in the same interrogation assay, toobtain the rapid screening for mutations that may be present. 11143 5′CGGAGCCTCCACCTCCCG SEQ ID NO: 216 CAH interrogator oligo 6 (wild type)for mutation site 1 11085 5′ CACCCTCCAGCCCCCAGC 3′ SEQ ID NO: 217 CAHinterrogator oligo 2 (pseudogene/mutant) for mutation site 2 11084 5′CGGAGCCTCCACCTCCTG 3′ SEQ ID NO: 218 CAH interrogator oligo 1(pseudogene/mutant) for mutation site 1 11086 5′ CCTCACCTGCAGCATCAAC 3′SEQ ID NO: 219 CAH interrogator oligo 3 (pseudogene/mutant) for mutationsite 3 11144 5′ CACCCTCCAGCCCCCAAC 3′ SEQ ID NO: 220 CAH interrogatoroligo 7 (wild type) for mutation site 2 11145 5′ CCTCACCTGCAGCATCATC 3′SEQ ID NO: 221 CAH interrogator oligo 8 (wild type) for mutation site 311087 5′ CCTGGAAGGGCACTT 3′ SEQ ID NO: 222 CAH interrogator oligo 4(pseudogene/mutant) for mutation site 4 11146 5′ CCTGGAAGGGCACGT 3′ SEQID NO: 223 CAH interrogator oligo 9 (wild type) for mutation site 411088 5′ GATTCAGCAGCGACTGTA 3′ SEQ ID NO: 224 CAH interrogator oligo 5(pseudogene/mutant) for mutation site 5 11147 5′ GATTCAGCAGCGACTGCA 3′SEQ ID NO: 225 CAH interrogator oligo 10 (wild type) for mutation site 511287 5′ CGAGGTGCTGCGCCTGCG 3′ SEQ ID NO: 226 CAH interrogation oligo 11(wild type) for mutation site 6 11288 5′ CGAGGTGCTGCGCCTGTG 3′ SEQ IDNO: 227 CAH interrogation oligo 12 (pseudogene/mutant) for mutation site6 11641 5′ GGGATCACATCGTGGAGATG 3′ SEQ ID NO: 228 CAH interrogationoligo 23 (wild type) for mutation site 7 11642 5′ GGGATCACAACGAGGAGAAG3′ SEQ ID NO: 229 CAH interrogation oligo 24 (pseudogene/mutant) formutation site 7

EXAMPLE 58 Amplification-Refractory Mutation System (ARMS) Followed byInterrogation

This Example further illustrates the detection of nucleic acid producedin ARMS reactions [Newton, C. R. et al. Nucleic Acid Res., 17:2503,(1989)] without running a gel to interpret results. ARMS is based on aPCR probe with a 3′ end mismatch at a site of mutation.

In this Example, PCR products are either made or not made with aparticular probe depending on the absence or presence of a single base(SNP site) in the target. The probe with a 3′-terminal mismatch cannotamplify the product on the mutant target but can produce a product onthe matched wild type target. In this example, unique restriction enzymesites are built into the PCR probe next to the Pst I restriction sitealready present in the probes so that the wild type and the mutantproducts are uniquely identified by the restriction site incorporated.11310 5′ GCTTAAGCTGCAGGGCATATGTGGTGATGATATCGTGGGTGAGTTC ATTTA 3′ SEQ IDNO: 230 11311 5′ GCTTAAGCTGCAGGGCCATGGTGGTGATGATATCGTGGGTGAGTT CATTTT 3′SEQ ID NO: 231 11284 5′ CTGGAAAATGAACTCACCCACGATATCATCACCA 3′ SEQ ID NO:232 11253 5′ AGCTTGGTGATGATATCGTGGGTGAGTTCATTTTCCAGGTAC 3′ SEQ ID NO:233 11255 5′ CTGGTAAATGAACTCACCCACGATATCATCACCA 3′ SEQ ID NO: 234 112545′ AGCTTGGTGATGATATCGTGGGTGAGTTCATTTACCAGGTAC 3′ SEQ ID NO: 235

Oligonucleotides named 11253 and 11254 were designed and cloned intoPromega's pGEM-7zf vector that had been cut with Kpn I and HinD IIIrestriction enzymes. These oligonucleotides contain a short region witha SNP. Clone 53 contains the sequences of oligonucleotides 11284 and11253. Clone 54 contains the sequences of oligonucleotides 11255 and11254. The sequences of clones 53 and 54 were confirmed by sequencing.Clones 53 and 54 have a unique EcoR V restriction site that provides ablunt end on cleavage. Clones 53 and 54 were digested to completion withSca I and then diluted to 1 ng/μL with water prior to use in thefollowing PCR reactions. PCR Master Mix:  50 μL 10 × Thermophilic buffer 20 μL 25 mM MgCl₂  10 μL 10 mM each dNTP  10 μL 100 μg/mL oligo 11314390 μL water

The PCR reactions were set up as follows. Master Plasmid Probe ReactionMix (1 μL) (1 μL) PCR-1 48 μL 53(WT*) 11311(WT) PCR-2 48 μL 53(WT)11310(Mut*) PCR-3 48 μL 54(Mut) 11311(WT) PCR-4 48 μL 54(Mut) 11310(Mut)*WT = wild type; Mut = mutant

The oligonucleotides used for PCR amplification were 11310 and 11311.Each of those oligonucleotides contains the Pst I restriction enzymesite, as does oligonucleotide 11314. The PCR cycling parameters usedwere 95° C., 6 minutes. After 4.5 minutes, 1 μL (5u/μL) of Taq DNApolymerase was added, and the reaction mixture was cycled (95° C., 30seconds; 60° C., 2 minutes)×30; 72° C., 5 minutes.

The four PCR products were digested in standard Pst I restriction enzymereactions, cleaned with the Wizard® PCR DNA Purification System (PromegaA7170), and eluted into 50 μL of water. These Pst I-digested PCRproducts were then cut with either no enzyme, Nco I or Nde I in standardrestriction enzyme digest reactions. Reaction 1 should cut with Nco I,but not with Nde I. Reaction 4 should cut with Nde I, but not with NcoI.

The following Interrogation Master Mix was assembled.   60 μL 10 × DNAPolymerase buffer  7.5 μL 40 mM NaPPi   15 μL 10 U/μL Klenow exo-    6μL  1 U/μL NDPK    6 μL 10 μM ADP 505.5 μL water

The following interrogation reactions were assembled, and incubated at37° C. for 1 hour. Then 1 μL of the reactions was combined with 19 μL ofinterrogation master mix (in duplicate), incubated for 15 minutes at 37°C. and 5 μL were added to 100 μL of L/L reagent and relative light units(rlu) were measured in a Turner® TD20/20 luminometer. The resultsobtained are shown below. Second Avg. Reaction cut DNA digest rlu 1.PCR-1 none 39.1 2. PCR-1 Nco I 149.1 3. PCR-1 Nde I 44.6 4. PCR-2 none26.5 5. PCR-2 Nco I 23.9 6. PCR-2 Nde I 25.2 7. PCR-3 none 28.4 8. PCR-3Nco I 26.8 9. PCR-3 Nde I 27.0 10.  PCR-4 none 43.4 11.  PCR-4 Nco I54.8 12.  PCR-4 Nde I 116.7

PCR-1 is activated for detection by close to four fold by digestion withNco I but is not activated by digestion with Nde I. PCR-4 is notsubstantially activated by digestion with Nco I, but is activated closeto 3 fold by digestion with Nde I. Neither PCR-2 nor PCR-3 was activatedby either enzyme, indicating the absence of product as anticipated. SEQID NO: 230 11310 5′ GCTTAAGCTGCAGGGCATATGTGGTGATGATATCGTGGGTGAGTTC ATTTA3′ SEQ ID NO: 231 11311 5′ GCTTAAGCTGCAGGGCCATGGTGGTGATGATATCGTGGGTGAGTTCATTTT 3′ SEQ ID NO: 232 11284 5′ CTGGAAAATGAACTCACCCACGATATCATCACCA 3′SEQ ID NO: 233 11253 5′ AGCTTGGTGATGATATCGTGGGTGAGTTCATTTTCCAGGTAC 3′SEQ ID NO: 234 11255 5′ CTGGTAAATGAACTCACCCACGATATCATCACCA 3′ SEQ ID NO:235 11254 5′ AGCTTGGTGATGATATCGTGGGTGAGTTCATTTACCAGGT AC 3′

EXAMPLE 59 Restriction Enzyme Digestion Prior to Interrogation

This Example concerns the use of two synthetic targets generated by PCRamplification with Pst I restriction enzyme sites on the probes. Thesetargets are first digested with Pst I, which leaves 3′ overhangs on bothends, making the products refractory to detection by means ofinterrogation using a Klenow exo− enzyme. The two PCR products differ inthe presence of another unique restriction enzyme site. The endsgenerated by this second digest provide ends that have a 5′ overhang andare permissive for interrogation.

Oligonucleotides 11315 (SEQ ID NO:236) and 11314 (SEQ ID NO:237) bothcontain a Pst I restriction enzyme site (CTGCAG) near the 5′ endpreceded by six arbitrary bases to permit efficient digestion. Theseoligonucleotides were used to create PCR products, about 200 base pairsin size, using the vectors pGEM-7zf(+) and pGEM-9zf(−) (Promega, P2251and P2391, respectively) for targets and using standard PCR conditions.

Forty microliters (800 ng) of each PCR product were digested with Pst Irestriction enzyme to completion and again purified with Wizard™ PCR DNAPurification System to remove the small digested fragment. Then the twodifferent PCR products were further digested in separate reactions withBamH I, Spe I and EcoR I enzymes or with no additional enzyme. BamH Idigests only the PCR product from pGEM-7zf(+) (PCR-1); Spe I digestsonly the PCR product from pGEM-9zf(−) (PCR-2); and EcoR I digests bothPCR products. All three of these enzymes leave a 5′ overhang that isresponsive to interrogation.

Four microliters of the Pst I digested DNA was digested with the secondenzyme, diluted two fold, and one microliter was combined with 19 μLmaster mix and incubated for 15 minutes at 37° C. Five microlitersthereafter were added to 100 μL of L/L reagent and relative light units(rlu) read on a Turner® TD20/20 luminometer to provide the data (induplicate) in the table below, thereby illustrating interrogations forthe presence or the absence of the second digestion. Master Mix: 10 ×DNA polymerase buffer  40 μL 40 mM Sodium Pyrophosphate  5 μL 10 U/μLKlenow exo- polymerase  10 μL  1 U/μL NDPK  4 μL 10 μM ADP  4 μLNanopure water 337 μL

Second enzyme Avg. Reaction DNA digestion rlu 1. PCR-1 none 38.9 2.PCR-1 BamH I 325 3. PCR-1 Spe I 62.2 4. PCR-1 EcoR I 386.9 5. PCR-2 none26.4 6. PCR-2 BamH I 37.3 7. PCR-2 Spe I 265.3 8. PCR-2 EcoR I 302.2

The PCR-1 product contains a BamH I and an EcoR I site, but no Spe Isite and responds appropriately. The PCR-2 product contains a Spe I andan EcoR I site, but no BamH I site and also responds appropriately.

These data demonstrate that detection was stimulated about 10 fold bydigestion with the appropriate enzyme. Furthermore, multiplexing wassimulated by mixing the two PCR fragments together and detecting onlyone of them by digestion with the appropriate enzyme followed byinterrogation. This method can thus be used to detect a singlenucleotide polymorphism that destroys or creates a restriction sitewithout running a gel. 11315 5′ ATGATGCTGCAGCAGGAAACAGCTATGAC 3′ SEQ IDNO: 236 11314 5′ ATGATGCTGCAGGTTTTCCCAGTCACGAC 3′ SEQ ID NO: 237

EXAMPLE 60 Detection of Rolling Circle Amplification Products Obtainedafter Circularization by Ligation

In this Example, a probe (oligonucleotide 10367 (SEQ ID NO:238)) ishybridized to either no target, or to a wild type (WT; 8831 (SEQ IDNO:239)) or a mutant (10354 (SEQ ID NO:240)) target and ligated into acircle. An extension probe (10368 (SEQ ID NO:241)) is then hybridizedand extended into run-around products, thereby amplifying the target.These products are detected using a complementary probe (10369 (SEQ IDNO:242)) in an interrogation reaction, with relative light unit outputbeing used to distinguish among the possible results. In order to obtaina low, no target, background, it is beneficial to treat the ligationreaction mixture with a combination of exonucleases to remove any DNAthat is not circular.

It is seen that the no target reaction gives very low light units andthere is about a six fold discrimination between WT and Mutant targets.In these studies, only a single rolling circle extension probe is usedand amplification is linear, not exponential. The basis for theWT/Mutant discrimination is whether or not the probe ligates into acircular molecule, because ligation is inefficient at a mismatchposition.

One microliter (500 ng) of oligonucleotide 10367 was combined with 1 μL(500 ng) of either oligonucleotide 8831 or 10354 or no target in threeseparate tubes. Water was added to a final volume of 8 μL. The solutionswere heated to 95° C. for 3 minutes, then cooled for 10 minutes at roomtemperature. One microliter of 10× E. coli ligase buffer (NEB) and 1 μL(10 u) E. coli ligase (NEB) were added. The solutions were furtherincubated for 60 minutes at 37° C. To each solution were then added: 0.5μL 50 U/μL T7 Gene 6 exonuclease (USB E700254) 0.5 μL 10 U/μLexonuclease I   2 μl 10 × Thermo Polymerase buffer   7 μL water

The solutions were then incubated for 15 minutes at 37° C., followed by10 minutes at 95° C. To each solution were then added:

-   1 μL 500 ng/μL 10368 (extension probe (SEQ ID NO:241))-   2 μL 2 mM dNTPs

The solutions were heated at 95° C. for 3 minutes, and then cooled for10 minutes at room temperature to anneal the extension probe. Onemicroliter (8 U) of Bst (LF) DNA polymerase was added and the tubes wereincubated at 42° C. for 10 minutes, then 65° C. for 30 minutes to permitextension. One unit of shrimp alkaline phosphatase was added and thetubes were incubated at 37° C. for 30 minutes, and then 65° C. for 15minutes.

To proceed with interrogation, the solutions were diluted 1:100 withwater and 1 μL of each of the three reactions was combined with 1 μL(500 ng) of interrogation oligonucleotide 10369 and 18 μL water. Eachcomposition so formed was heated at 95° C. for 3 minutes, then cooledfor 10 minutes at room temperature. Twenty microliters of master mixwere added, and each solution was incubated at 37° C. for 15 minutes.Then, 4 μL of each solution were combined with 100 μL of L/L reagent induplicate, and light output read in a Turner® TD20/20 luminometer. Theresults obtained were as shown below. Sample average rlu No target 5.8WT target 248.6 Mutant target 43.5

SEQ ID NO: 238 10367 5′ ACAACGTCGTGACTAGGATCACGCTAATGCTTCAGCCTGATGAGTCCGATCAGCCTGATGAGTCCGATCTGGCCGTCGTTTT 3′ (circle probe) SEQ ID NO: 2398831 5′ CAGTCACGACGTTGTAAAACGACGGCCAGT 3′ (WT target) SEQ ID NO: 24010354 5′ CAGTCACGACGTTGTGAAACGACGGCCAGT 3′ (mutant target) SEQ ID NO:241 10368 5′ AGCATTAGCGTGATCC 3′ (rolling circle extension probe) SEQ IDNO: 242 10369 5′ CAGCCTGATGAGTCCG 3′ (circle interrogation probe)

EXAMPLE 61 Detection of Rolling Circle Amplification Products of M13 DNA

This Example uses the pUC/M13 Forward primer (17mer, Promega Q5391) tohybridize to single stranded (ss) M13mp18 DNA and synthesize rollingcircle DNA using Bst LF (large fragment) thermostable DNA polymerase.The free nucleotides are then removed with Shrimp Alkaline Phosphatase(SAP) and the rolling circle products detected by pyrophosphorolysis ofthe probe that is complementary to this product, pUC/M13 primer, reverse(17mer) Promega Q5401.

The following reactions were assembled in duplicate: M13mp18 10 × Buffer2 μM Probe Water Sample DNA (μL) (μL) dNTPs (μL) (μL) (μL) 1 1 (250 ng)2 2 none 14 2 1 (250 ng) 2 2 5 9 3 1 (250 ng) 2 2 5 9

The assembled solutions were incubated at 95° C. for 3 minutes, thencooled for 10 minutes at room temperature.

To samples 1 and 3, was added 1 μL (8 U) Bst LF DNA polymerase, and alltubes were incubated for 30 minutes at 65° C., then cooled for 2 minuteson ice. One unit of Shrimp Alkaline Phosphatase was added, the solutionsincubated at 37° C. for 30 minutes, then heated to 65° C. for 15 minutesto denature the phosphatase enzyme.

One microliter of the reactions was added to 5 μL (50 ng) of pUC/M13reverse primer, the interrogation oligonucleotide probe, and heated to95° C. for 3 minutes, then cooled for 10 minutes at room temperature.Twenty microliters master mix were added, and the tubes were heated at37° C. for 15 minutes. Four microliters were then combined with 100 μLof L/L Reagent, and the light output read on a Turner® TD20/20luminometer. Reaction Avg. rlu 1. (no extension probe) 3.5 2. (no Bst LFDNAP) 4.1 3. (complete reaction) 68.7

It can be seen that a signal of about 20 times that of the controls isdependent on the presence of both the Bst (LF) DNA thermopolymerase andthe rolling circle extension probe (forward probe).

EXAMPLE 62 Ligase Chain Reaction Prior to Interrogation

In this Example, Ligase Chain Reaction (LCR) was performed to amplifywild type and mutant species of the lacI gene fragment used as anamplification-control sequence in the LCR kit (Stratagene, 200520)followed by interrogation. LCR is a DNA amplification technique thatutilizes a cyclic two-step reaction. Target DNA is denatured at anelevated temperature followed by the annealing of two sets ofcomplementary oligonucleotides to the denatured DNA and their ligationwith a thermostable ligase. The ligation products from one cycle serveas targets for the next cycle's ligation reaction.

Oligonucleotide 11192 (SEQ ID NO:243) is complementary to 11195 (WT)(SEQ ID NO:244) and 11196 (mutant) (SEQ ID NO:245). Oligonucleotide11197 (SEQ ID NO:246) is complementary to 11193 (WT) (SEQ ID NO:247) and11194 (mutant) (SEQ ID NO:248). The mutant oligonucleotide differs fromits counterpart wild type oligonucleotide only at the 3′-terminal base.

The wild type and mutant targets that are present in the LCR kit wereused as the targets for the LCR reaction performed according to kitinstructions. The LCR product was then quantified and used as the targetfor interrogation. One microliter of the wild type LCR target (100 pg)was combined with 1 μg of wild type interrogation oligonucleotide (11198(SEQ ID NO:249)), mutant interrogation oligonucleotide (11199 (SEQ IDNO:250)), or no oligonucleotide along with water to a final volume of 20μL. A target-only reaction and a probe-only reaction were also assembledas controls. Likewise, similar solutions were assembled with the mutantLCR target. The solutions were heated at 95° C. for 8 minutes todenature the nucleic acid. LCR was performed according to manufacturer'sinstruction.

The following master mix was assembled. 432 μL water 120 μL 10 × DNA polbuffer (Promega, M195A)  15 μL 40 mM NaPPi  15 μL Klenow exo- (1 u/μL) 6 μl NDPK 1 U/μL  12 μL ADP 10 μM

Twenty microliters of master mix were added to each solution, and thesolutions were further incubated at 37° C. for 15 minutes. Fivemicroliters of each solution were then combined with 100 μL of L/Lreagent and light output was measured on a Turner® TD20/20 luminometer.The results of average relative light units (Avg. rlu) are shown below:Avg. rlu Wild type target rxns Probe 11198 (WT) + target 58.35 Probe11199 (mutant) + target 5.29 Probe 11198 only 2.77 Target only 3.89Mutant target rxns Probe 11198 (WT) + target 7.78 Probe 11199 (mutant) +target 77.22 Probe 11199 only 3.29 Target only 7.05

The data indicate that about 10-fold match/mismatch discrimination canbe obtained when performing the interrogation reaction after an LCRamplification reaction. 11192 5′ TTGTGCCACGCGGTTGGGAATGTA 3′ SEQ ID NO:243 11195 5′ TACATTCCCAACCGCGTGGCACAAC 3′ SEQ ID NO: 244 11196 5′TACATTCCCAACCGCGTGGCACAAT 3′ SEQ ID NO: 245 11197 5′AACTGGCGGGCAAACAGTCGTTGCT 3′ SEQ ID NO: 246 11193 5′AGCAACGACTGTTTGCCCGCCAGTTG 3′ SEQ ID NO: 247 11194 5′AGCAACGACTGTTTGCCCGCCAGTTA 3′ SEQ ID NO: 248 11198 5′ TTTGCCCGCCAGTTGTT3′ SEQ ID NO: 249 11199 5′ TTTGCCCGCCAGTTATT 3′ SEQ ID NO: 250

EXAMPLE 63 Detection of Chromosomal DNA Without Amplification: I

In theory, direct detection of a single copy gene in chromosomal DNAshould be possible if enough DNA can be assayed. The amount of humangenomic DNA needed can be calculated as follows:$\frac{\left( {1 \times 10^{- 9}\quad g\quad{DNA}} \right)\left( {5 \times 10^{9}\quad{{bases}/{genome}}} \right)}{\left( {1 \times 10^{3}\quad{bases}\quad{specific}\quad{target}} \right)} = {{approximately}\quad 5\quad{mg}\quad{of}\quad{DNA}}$However, as the amount of DNA interrogated increases, nonspecific DNAsignal from this DNA also increases. Therefore, chromosomal DNA inamounts approaching even 1 μg of DNA would produce very high background.

Increasing the copies of target DNA per chromosome is one way toovercome this limitation. Many such sequences are known. The absolutesequence of the repeated DNA in different species can vary as does thenumber of copies of the sequence in the genome. For example, there areestimated to be 500-1000 copies of a sequence known as the rep sequencein the E. coli chromosome. The Alu sequence is present in the haploidhuman chromosome in approximately 300,000 copies. The estimated amountof human chromosomal DNA needed to detect the Alu sequence is:$\frac{5 \times 10^{- 3}\quad{grams}\quad{{DNA}\left( {{single}\quad{copy}\quad{gene}\quad{requirement}} \right)}}{3 \times 10^{5}\quad{copies}\quad{per}\quad{genome}} = {1.7 \times 10^{- 8}\quad{{grams}\left( {{or}\quad{about}\quad 17\quad{ng}\quad{of}\quad{DNA}} \right)}}$In this example, probes to two regions of the Alu sequence (Alu 1oligonucleotide 11597 (SEQ ID NO:251) and Alu 2 oligonucleotide 11598(SEQ ID NO:252)) were used to demonstrate that direct detection ofchromosomal DNA is achievable.

The genomic DNA (4.2 μg) was digested to completion (5 hours, 37° C.)with 40 units of Sph I restriction enzyme, which leaves a 3′ overhang onthe digested fragments. Either 40 ng or 80 ng of the digested genomicDNA was annealed to 1.0 μg of the interrogation probes 11597 and 11598in separate reactions, and 11597 and 11598 in the same reaction withwater added to a final volume of 20 μL. A negative control, without aninterrogation probe, was also assembled. The solutions were heated at92° C. for 3 minutes and cooled at room temperature for 15 minutes.

Twenty microliters of master mix, described below, were added to eachannealing reaction and the tubes were further incubated at 37° C. for 20minutes, then stored on ice. Four microliters of the reaction were addedto 100 μL of L/L reagent (Promega F120B) in quadruplicate samples, andrelative light units (rlu) measured on a Turner® TD20/20 luminometer.The rlu results are reported below. Master Mix: 200 μL 10 × DNAPolymerase Buffer  25 μL 40 mM NaPPi  25 μL Klenow exo-  10 μL NDPK 1U/μL  20 μL ADP 10 μM 720 μL water

Rxn* 1 Rxn 2 Rxn 3 Rxn 4 average Net Std Dev* No DNA 3.372 3.342 3.3063.249 3.317 0 0.052898 alu1 only 3.92 3.625 3.756 3.799 3.775 0.4580.12174 alu2 only 23.18 25.47 24.58 25.19 24.61 21.29 1.020082 40 ng DNA20.63 21.98 23.91 22.3 22.21 18.39 1.347504 alu1 + 40 ng 53.12 57.0552.52 36.5 49.80 46.03 9.089798 DNA alu2 + 40 ng 99.23 91.26 55.9 85.5983.00 58.39 18.90995 DNA 80 ng DNA 38.57 44.34 42.96 46.33 43.05 39.733.291454 alu1 + 80 ng 89.25 68.01 91.43 96.14 86.21 82.44 12.46776 DNAalu2 + 80 ng 156.2 156.6 149.9 143.7 151.6 127.0 6.095353 DNA alu1 +alu2 30.65 23.82 32.57 27.60 28.66 25.34 3.820881 alu1 + alu2 + 40 ng66.49 101.1 104.9 104.3 94.1975 65.81 18.54682 DNA*Std Dev = 1 standard deviation, Rxn = reaction

11597 5′ AGACCCCATCTCTAA 3′ SEQ ID NO: 251 (Alu 1) 11598 5′GCCTGGGTGACAGAGCA 3′ SEQ ID NO: 252 (Alu 2)

EXAMPLE 64 Detection of Chromosomal DNA Without Amplification: II

Another method to detect the presence of the Alu sequence is to performsingle probe extension reactions as described in Example 50. Probes aredesigned to bind to a target Alu sequence and be extended. Followingextension, the probe can form a “hairpin” structure—forming a stretch ofdouble strand DNA. This DNA is then detected in a “probeless”pyrophosphorylation assay. If the extended probe sequence extends beyondthe segment of the probe designed to form one segment of the hairpin,the product is not expected to be detected because the product has a 3′overhang. In order to prevent such a situation, the probes that havebeen designed can be used in reactions missing one of the four DNAbases. By performing the reactions in this way, the probe is notextended beyond the region of hybridization. Scheme 1 illustrates howtwo such probes hybridize to an Alu sequence.

The predicted extended products of these probes and the secondarystructure of the hairpins that the extended products can form are shownin Scheme 2, below. Scheme 2 Genbank#AF085897 Extension of oligo A:5′CTCCAGCCTCGGTGACAGAGCAAGACCCTGTCTCAAAAAAAAA

Hairpin Secondary structure:

Genbank#AL022238 Extension of oligo B:5′CTCCAGCCTGAGCAACACAGCAAGACCCTGTCTCAAAACAAAAC

Hairpin Secondary structure:

Oligo A 5′CTCCAGCCTCGGTGACAGAGCAAGACCCTGTCTCAAAAAAAAAGCCTCGGT SEQ IDNO:253 GCAGGGTCTTGCTCTGT 3′ Oligo B5′CTCCAGCCTGAGCAACACAGCAAGACCCTGTCTCAAAACAAAACGCCTGAG SEQ ID NO:254CCAGGGTCTTGCTGTGTT 3′

EXAMPLE 65 HPLC Separation of dNTPs after Interrogation Assay, but Priorto Phosphate Transfer and Light Production

Large-volume pyrophosphorylation assays were performed on matched andmismatched probe/target hybrids. The released nucleotides were separatedby high performance liquid chromatography (HPLC) and their fractionscollected. NDPK terminal phosphate transfer reactions were performed onthese concentrated fractions and luciferase assays conducted toillustrate discrimination between the original matched and mismatchedhybrid treated samples.

Target/probe hybrids were formed by combining 315 ng of the syntheticwild type CMV target oligonucleotide with either 10.5 μg wild type CMVprobe for a matched hybrid, or 10.5 μg mutant CMV probe for a mismatchedhybrid, and adding water to a final volume of 200 μL. Theoligonucleotides were CV 12 (SEQ ID NO:9), CV 15 (SEQ ID NO:12), and CV16 (SEQ ID NO:13), as previously described in Example 2. These solutionswere heated to 95° C. for at least 5 minutes, then cooled at roomtemperature for at least 10 minutes.

The following master mix was prepared. 337.5 μL Nanopure water (Promega,AA399)  90.0 μL 10 × DNA Polymerase buffer (Promega, M195A) 11.25 μL 40mM NaPPi (Promega, C113)

Master mix (210 μL) was added to each of the above hybrid solutions and5.8 units of Klenow exo− (Promega, M218A) were added to each. Thesolutions were then incubated at 37° C. for 15 minutes and stored onice. HPLC separation of the dNTPs was performed.

HPLC separation of dATP, dCTP, dGTP and TTP was performed on a 100×4.6mm, 3μ Luna C18 column [Perrone and Brown, J. Chromatography,317:301-310 (1984)] from Phenomenex. The column was eluted with a lineargradient of 97 percent buffer A (100 mM triethylammonium acetate, pH 7)to 92 percent buffer A over a period of 12 minutes. The composition ofbuffer B is 80:20 acetonitrile:35 mM triethylammonium acetate. Detectionwas monitored by absorbance at 250, 260 and 280 nm. Under theseconditions, dCTP was found to elute between 4 and 4.5 minutes, TTP anddGTP eluted as two peaks between 7 and 7.5 minutes, and dATP eluted from9 to 9.5 minutes.

The fractions containing the free dNTPs were collected and lyophilized.Fraction one contained dCTP, fraction two contained dGTP and TTP, andfraction three contained dATP.

Each fraction was reconstituted in 100 μL of nanopure water. Tenmicroliters of each fraction, or 10 μL of water as a control, were addedto a 10 μL mixture of water, 10× DNA Polymerase Buffer, and ADP so thatthe final concentration was 1× DNA pol buffer and 0.1 μM ADP. NDPK(0.005 units) was added to each tube in one set of the tubes and anequal amount of water was added to each tube in the other set of tubes.Samples and controls were incubated at 37° C. for 15 minutes, 10 μLadded to 100 μL of L/L reagent and the light output was measured on aTurner® TD10/20 luminometer. The relative light units (rlu) resultsobtained are shown below: Avg Sample Trial 1 Trial 2 Trial 3 rlu Matchedhybrid with NDPK Fraction 1 206.6 200.6 205.9 204.4 Fraction 2 839.4851.6 833.9 841.6 Fraction 3 1149.0 1150.0 1169.0 1156 Mismatched hybridwith NDPK Fraction 1 101.8 97.0 98.9 99.9 Fraction 2 386.1 387.3 382.2385.2 Fraction 3 412.4 409.9 416.5 412.9 Match hybrid without NDPKFraction 1 6.8 6.5 — 6.6 Fraction 2 10.9 11.5 — 11.2 Fraction 3 33.037.8 — 35.4 Mismatched hybrid without NDPK Fraction 1 6.2 6.7 — 6.4Fraction 2 8.3 8.4 — 8.4 Fraction 3 13.4 13.5 — 13.4 No dNTP 7.9 7.5 —7.7

As is seen from the above data, the fraction one match:mismatch ratio is2.1, fraction 2 match:mismatch ratio is 2.2 and fraction 3match:mismatch ratio is 2.8. The data therefore demonstrate the utilityof using HPLC separation of individual nucleotides followed by NDPKconversion to ATP, the preferred substrate of luciferase. Fraction 3provides a slightly higher match:mismatch ratio owing to the presence ofdATP in the nucleotide HPLC fraction. Nevertheless, HPLC separation ofidentifier nucleotides is useful in the interrogation assays of thepresent invention. CV12 5′CCAACAGACGCTCCACGTTCTTTCTGACGTATTCGTGCAGCATGGTCTGCG SEQ ID NO: 9AGCATTCGTGGTAGAAGCGAGCT 3′ CV15 5′ CTACCACGAATGCTCGCAGAC 3′ SEQ ID NO:12 CV16 5′ CTACCACGAATGCTCGCAGAT 3′ SEQ ID NO: 13

EXAMPLE 66 Mass Spectrometry for Nucleotide Detection

The mass spectrometer uses the ratio of molecular mass to charge ofvarious molecules to identify them. Nucleic acids are made up of fourdifferent base molecules, each with a different mass to charge ratio. Inthis Example, the capability to use mass spectrometry for separation ofthe nucleotides that make up DNA is demonstrated.

The ESIMS (Electro Spray Ion Mass spectrometry) spectra of 1 μM and 0.1μM NTP molecules were determined (Fisons Instruments, VG Platform). Thesamples were prepared by diluting 1:1 with acetonitrile/water/1% NH₄OH.A 20 μL injection was made for each sample. Therefore, 10 picomoles ofeach dNTP are in the 1 μM sample injection, and 1 picomole of each dNTPis in the 0.1 μM sample injection.

Each of the dNTPs is observed in the 1 μM sample along with the dNTP+Na⁺peaks. There was a 485 peak also present, which is an impurity in thesystem or samples. The peaks for each of the dNTPs are significantlydiminished in the 0.1 μM sample; only the dATP peak is above the noiselevel. Therefore, the difference between the 1 and 0.1 μM samples can bequalitatively determined, which indicates the ability to determine thedifference between interrogation samples in which the probe and targetare matched and mismatched at the 3′-terminal region of the probe.

EXAMPLE 67 Speciation-Detection of Mitochondrial DNA Specific to VariousAnimals

In this example, a segment of mitochondrial DNA comprising a segment ofthe cytochrome B gene was amplified from a variety of animal speciesusing PCR primers 11590 (SEQ ID NO:255) and 11589 (SEQ ID NO:256) (PNAS86:6196-6200). These PCR primers were diluted in 10 mM Tris, pH 7.5, toa final concentration of 0.22 μg/μL. The genomic DNAs used were bovine(Clontech, 6850-1), chicken (Clontech, 6852-1), dog (Clontech, 6950-1)and human (Promega, G1521).

The PCR reactions were assembled to include 5 μL 10× buffer with 15 mMMgCl₂ (Promega, M188J), 1 μL dNTPs 10 mM (Promega, C144G), 2 μL primer11590, 2 μL primer 11589, 0.5 μL Taq polymerase 5 U/μL (Promega, M186E),and 38.5 μL water. To each tube was then added 1 μL (100 ng) of genomicDNA. The PCR cycling parameters were (15 seconds, 94° C.; 15 seconds,55° C.; 30 seconds, 72° C.)×30. The size of PCR products was confirmedby running an aliquot on an agarose gel and visualizing with ethidiumbromide (EtBr) staining. The PCR products were then separated from freenucleotides (Promega, A7170) and an aliquot run on an agarose gel. Allsamples produced a PCR product of the same size.

Each PCR DNA was then used in an assay to determine if it could bespecifically identified with a species-specific probe. One microliter ofinterrogation probe (1 μg/μL) and 17 μL water were combined with 2 μL ofthe appropriate PCR product and heated at 91° C. for 3 minutes, thencooled at room temperature for 15 minutes. Twenty microliters of mastermix (described below) were added to each tube and each was furtherincubated at 37° C. for 15 minutes. Four microliters of the solutionswere then added to 100 μL L/L reagent (Promega F120B), and the relativelight output (rlu) measured on a Turner® TD20/20 luminometer. The rluaverage values from two reactions, minus the DNA background values,along with the standard deviation values are listed below. Master mix:  312 μL 10 × DNA pol buffer (Promega M195A)   39 μL NaPPi 40 mM(Promega E350B)   39 μL Klenow exo minus (Promega M128B)  15.6 μL NDPK 1U/μL  31.2 μL ADP 10 μM (Sigma)  1123 μL water (Promega AA399)

Averages from 2 reactions. Net light units are calculated by subtractingthe DNA background Standard Deviations No Human Chicken Cow Dog No HumanChicken Cow Dog Probe DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA comzoo−0.096 44.5 14.25 119.7 124.6 0.654 7.000 23.33 3.465 8.63 huzoo1 1.77138 −40 −51.85 −63.55 0.137 38.96 31.74 6.505 12.52 huzoo2 −0.889 101.6−23.35 −0.05 −48.75 0.761 3.959 0.141 8.768 2.19 chzoo1 43.07 −30.434.05 −2.75 −31.15 7.078 1.909 6.364 2.687 8.70 chzoo2 −0.361 57.6 50.733.05 −3.25 0.075 43.77 29.34 12.59 21.43 cozoo2 1.925 90.95 125.1 202.6132.5 0.208 20.08 13.22 8.627 19.30 dozoo2 0.966 71.8 158.7 0.180 9.5461.98

The data demonstrate that the primers detect the mitochondrial PCRproduct. Both of the human-specific probes (11576 (SEQ ID NO:257)) and11583 (SEQ ID NO:258)) were shown to be specific for human mitochondrialDNA. The common probe, 11582 (SEQ ID NO:259), detected all of thespecies, but was less efficient with chicken DNA. The chicken-specificprobe, 11577 (SEQ ID NO:260), was specific for chicken mitochondrialDNA, but the other chicken-specific probe, 11584 (SEQ ID NO:261),detected all the species except dog. The cow-specific probe, 11588 (SEQID NO:262), gave the best detection signal for cow DNA, but alsodetected the other species. The dog-specific probe, 11586 (SEQ IDNO:263), was assayed only with dog and cow DNA, but detected the dog DNAbetter than cow DNA. A cleaner PCR product provides DNA with lessbackground. SEQ ID NO: 255 5′ AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA 3′11590 zooamp2 SEQ ID NO: 256 11589 zooamp1 5′AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA 3′ 11576 huzoo1 5′ CCAGACGCCTCA 3′SEQ ID NO: 257 11583 huzoo2 5′ ACCTTCACGCCA 3′ SEQ ID NO: 258 11582comzoo 5′ TGCCGAGACGT 3′ SEQ ID NO: 259 11577 chzoo1 5′ GCAGACACATCC 3′SEQ ID NO: 260 11584 chzoo2 5′ GGAATCTCCACG 3′ SEQ ID NO: 261 11588cozoo2 5′ ACATACACGCAA 3′ SEQ ID NO: 262 11586 dozoo2 5′ ATATGCACGCAA 3′SEQ ID NO: 263

EXAMPLE 68 Trisomy Detection

Detection of a simulated trisomy sample is demonstrated in this Example.The nucleic acid probes and targets were previously described in Example49. These include the CMV probes 9211 (SEQ ID NO:86) and 9212 (SEQ IDNO:35), the p-ZERO-2 clone of the double-stranded synthetic CMV wildtype target CMV-A (10800 (SEQ ID NO:171) and 10801 (SEQ ID NO:172)), andthe p-ZERO-2 clone of the double-stranded synthetic CMV mutant targetCMV-G (10803 (SEQ ID NO:173) and 10805 (SEQ ID NO:174)).

Each p-ZERO-2 plasmid (1 μg) was digested to completion with Pst Irestriction enzyme at 37° C. for 1 hour. Ten microliters of the digestwere then further diluted with 20 μL of water.

The following Master Mix was assembled.   60 μl 10 × DNA polymerasebuffer (Promega M195A)  7.5 μl 40 mM NaPPi (Promega C113)  1.5 μl 10U/μL Klenow exo- (Promega M128A)   3 μL NDPK   6 μL 10 μM ADP (PromegaA5285)  225 μL water (Promega AA399)

The following solutions were assembled using the digested and dilutedtemplates. Simulated Solution Template Probe (μL) Water phenotype rlu 11 μL CMV-A 1 μL 9211 18 homoZ* A 53.27 2 1 μL CMV-A 1 μL 9212 18 homoZ A7.19 3 1 μL CMV-G 1 μL 9211 18 homoZ G 5.78 4 1 μL CMV-G 1 μL 9212 18homoZ G 63.84 5 1 μL CMV-A 1 μL 9211 17 heteroZ* 1:1 54.08 1 μL CMV-G 61 μL CMV-A 1 μL 9212 17 heteroZ 1:1 61.54 1 μL CMV-G 7 2 μL CMV-A 1 μL9211 16 trisomy 90.87 1 μL CMV-G 2:1 (A:G) 8 2 μL CMV-A 1 μL 9212 16trisomy 64.74 1 μL CMV-G 2:1 (A:G) 9 1 μL CMV-A 1 μL 9211 16 trisomy50.86 2 μL CMV-G 1:2 (A:G) 10 1 μL CMV-A 1 μL 9212 16 trisomy 111.0 2 μLCMV-G 1:2 (A:G)*homoZ = homozygous; heteroZ = heterozygous.

The solutions were heated at 95° C. for 3 minutes then cooled for 10minutes at room temperature. Then, 20 μl master mix were added, and thesolutions further heated at 37° C. for 15 minutes. Four microliters ofthe solutions were then added to 100 μL of L/L reagent (Promega F202A)and the relative light output measured immediately on a Turner® TD20/20luminometer. The rlu values are listed above.

The rlu values demonstrate that the 1:2 (A:G) template mix exhibits a1:2 rlu ratio, whereas the heterozygous 1:1 A:G rlu ratio is close to1:1. A contemplated method is thus shown to be useful in detectingtrisomy. CMV Interrogation oligos Wild Type 5′ SEQ ID NO: 86 CMV-ACACTTTGATATTACACCCATG 3′ Probe: (9211) Mutant CMV- 5′ SEQ ID NO: 35 Gprobe: CACTTTGATATTACACCCGTG 3′ (9212) 10800 5′ CGTGTATGCCACTTTG SEQ IDNO: 171 ATATTACACCCATGAACGTG CTCATCGACGTGAACCCGCA CAACGAGCT 3′ 10801 5′CGTTGTGCGGGTTCAC SEQ ID NO: 172 GTCGATGAGCACGTTCATGGGTGTAATATCAAAGTGGCAT ACACGAGCT 3′ 10803 5′ CGTGTATGCCACTTTG SEQ ID NO:173 ATATTACACCCGTGAACGTG CTCATCGACGTGAACCCGCA CAACGAGCT 3′ 10805 5′CGTTGTGCGGGTTCAC SEQ ID NO: 174 GTCGATGAGCACGTTCACGGGTGTAATATCAAAGTGGCAT ACACGAGCT 3′

EXAMPLE 69 Comparison of Thermophilic DNA Polymerases in a One-Step 70°C. Interrogation Reaction

In this example, four different thermophilic DNA polymerases were usedalong with the thermophilic NDPK from Pfu in an interrogation reaction.The polymerases used were Taq (Promega, M166F), Pfu (Pyrococcus furiosusstrain Vc1 DSM3638, Promega, M774A), Tvu (Thermoactinomyces vulgaris,purified at Promega), and Ath (Anaeocellum thermophilum, purified atPromega).

Cytomegalovirus (CMV) synthetic targets were generated by combining wildtype oligonucleotide primers 9162 (SEQ ID NO:31) and 9165 (SEQ ID NO:32)or mutant oligonucleotide primers 9163 (SEQ ID NO:33) and 9166 (SEQ IDNO:34). The interrogation oligonucleotides used were wild type sequence9211 (SEQ ID NO:86) and mutant sequence 9212 (SEQ ID NO:35).

Five nanograms of either the wild type or the mutant target (2.5 ng eachof 9162 and 9165 for wild type or 9163 and 9166 for mutant) werecombined with 1 μg of either the wild type probe, the mutant probe, orno probe, and water to a final volume of 20 μL. The solutions wereheated for 5 minutes at 95° C. then cooled for 10 minutes at roomtemperature. Twenty microliters of 2× master mix were then added to eachsolution, and each was further incubated at 70° C. for 10 minutes. Fourmicroliters of each solution were added to 100 μL of L/L Reagent(Promega F202A) and the relative light units (rlu) measured on a Turner®TD20/20 luminometer. The various combinations of target and probeassayed and their average resulting rlu values, corrected for backgroundvalues, from duplicate solutions are listed below. 2 × Master Mix: 100μL 10 × Thermophilic DNA polymerase buffer (Promega, M190A) 100 μL 15 mMMgCl₂ (Promega, A351B)  25 μL 40 mM NaPPi (Promega, E350B)  10 μL 10 μMADP (Sigma, A-5285)  5 μL Thermophilic polymerase (1 U enzyme/reaction) 5 μl Pfu NDPK (0.5 U/μL) (see Example 25 for enzyme purification; 0.1U/rxn)) 275 μL water

match:mismatch Polymerase Target Probe rlu ratio Taq wild type wild type129 128:1 wild type mutant −2 mutant mutant 62  95:1 mutant wild type0.65 Pfu wild type wild type 121  20:1 wild type mutant 6 mutant mutant34  1:2 mutant wild type 54 Tvu wild type wild type 898  89:1 wild typemutant 10 mutant mutant 1075  66:1 mutant wild type 16 Ath wild typewild type 327 327:1 wild type mutant 0 mutant mutant 244 136:1 mutantwild type 1.8

SEQ ID NO: 31 9162 5′ CGTGTATGCCACTTTGATATTACACCCATGAACGTGCTCATCGACGTCAACCCGCACAACGAGCT 3′ SEQ ID NO: 32 9165 5′CGTTGTGCGGGTTCACGTCGATGAGCACGTTCATGG GTGTAATATCAAAGTGGCATACACGAGCT 3′SEQ ID NO: 33 9163 5′ CGTGTATGCCACTTTGATATTACACCCGTGAACGTGCTCATCGACGTCAACCCGCACAACGAGCT 3′ SEQ ID NO: 34 9166 5′CGTTGTGCGGGTTCACGTCGATGAGCACGTTCACGG GTGTAATATCAAAGTGGCATACACGAGCT 3′SEQ ID NO: 86 9211 5′ CACTTTGATATTACACCCATG 3′ (wild type primer) SEQ IDNO: 35 9212 5′ CACTTTGATATTACACCCGTG 3′ (mutant primer)

EXAMPLE 70 Replicate Interrogations of One Target

This example demonstrates the amount of fluctuation when the same targetis interrogated with the same probe in 30 replicates of the reaction. Inthis example the PCR purification is automated on a Beckman Biomek™robot and the luciferase light output measurements are automated on anEG&G Berthold Microlumat Plus luminometer.

Ten 100 μL PCR reactions were assembled as follows.  10 μL 10 × PCRbuffer (Promega, M190A)   8 μL 25 mM MgCl₂   2 μL 10 mM dNTPs   2 μLFactor V probe 10861 (SEQ ID NO: 264), 50 pmol/μL   2 μL Factor V probe9828 (SEQ ID NO: 265), 50 pmol/μL   4 μL human genomic DNA (40 ng)  72μL water 2.5 U Taq polymerase (Promega, M186A)

The cycling parameters were 94° C., 2 minutes; (94° C., 30 seconds; 60°C., 1 minute; 70° C., 1 minute)×35; 70° C., 5 minute; 4° C. soak. Aftercycling, the ten reactions were pooled together and a 10 μL aliquot wasrun on a 1% agarose gel to confirm that the correct size product wasmade.

Twenty-five microliter aliquots of the PCR product were distributed intoeach of 32 wells of a Dynex™ 96 well plate. The PCR product was thenpurified from free nucleotides on a Beckman Biomek™ robot. Two hundredfifty microliters of MagneSil™ paramagnetic particles were resuspendedin 9 mL of a solution containing 0.4 M guanidine thiocyanate and 0.08 Mpotassium acetate. Of that suspension, 180 μL were added to each welland mixed. The samples were magnetized and the supernatant removed.

The particles were then washed three times with 100 μL of 70% ethanol.Then 50 μL of water and 150 μL of 0.4 M guanidine thiocyanate and 0.08potassium acetate were added to each well. The samples were magnetizedand the supernatant removed. Again, the particles were washed threetimes with 100 μL of 70% ethanol, air dried for 10 minutes and elutedinto 100 μL of water, which was transferred to a new plate.

The following master mix was assembled. 3905 μL water 1100 μL 10 × DNApolymerase buffer  275 μL 40 mM NaPPi  110 μL 10 μM ADP  55 μL  1 U/μLNDPK  55 μL 10 U/μL Klenow exo-Ten microliters of each purified PCR product aliquot were added to 3wells of a luminescence spectroscopy 96-well plate. To one of thereplicates were added 10 μL (150 pmol) of wild type probe 11505 (SEQ IDNO:266); to another of the replicate wells were added 10 μL (150 pmol)Factor V Leiden mutant oligonucleotide 11432 (SEQ ID NO:267); and to thethird well were added 10 μL of water. Sodium hydroxide (10 μL of 0.06N)was added to all wells as were 10 μL of 0.1 M Tris pH 7.3. The plate wasincubated at 37° C. for 5 minutes, and then 25 μL of master mix wereadded to each well. The plate was further incubated at 37° C. for 15minutes.

Then, 100 μL of L/L reagent (Promega, F202A) were added serially to eachof the wells. The light output was measured in a Berthold luminometerimmediately after the L/L reagent was added and before the robot addedthe L/L reagent to the next well. The relative light unit (rlu) resultsare provided below and demonstrate consistent values although someincrease is obtained that is reflective of the longer time that thereaction is at 37° C. with the master mix. Water WT Mutant No Interr.96273 26312 29132 113830 29400 30506 125410 30445 31932 121334 3004032347 136788 28943 30280 144237 34935 36664 161304 39844 41521 14297934612 37252 157255 42247 44432 156354 39103 38966 163757 40294 40302157427 39202 42753 163480 43519 44072 165872 47608 47560 163614 4250743241 158910 37910 39909 164992 46147 46845 168549 53255 52263 17247153922 52196 158835 40988 43717 169860 51393 52309 166303 46488 46870178970 60475 58484 167606 46088 50270 171895 50478 52369 174249 5655456536 180951 60283 54489 159357 37508 39647 176683 49491 51971 16851441909 44462

PCR probe sequences: 10861 5′ TGCCCAGTGCTTAACAAGACCA 3′ SEQ ID NO: 264

The first four linkages at the 5′ end are phosphorothioate linkages. 9828 5′ TGTTATCACACTGGTGCTAA 3′ SEQ ID NO:265 11505 5′GACAAAATACCTGTATTCCTCG 3′ SEQ ID NO:266 11432 5′ GACAAAATACCTGTATTCCTTG3′ SEQ ID NO:267

EXAMPLE 71 Multiplex Determination of Nucleotide Sequences Associatedwith Factor V Leiden and with a Prothrombin SNP in the Same Reaction

A assay is performed in this Example to determine if a human DNA samplecontains the Leiden mutation of the Factor V gene, as well as aparticular Prothrombin single nucleotide polymorphism (SNP). The assayfor these two characteristics is performed simultaneously in the samereaction.

Probes PT5 (SEQ ID NO:104) and PT6 (SEQ ID NO:105) were used toPCR-amplify a region of human genomic DNA spanning about 500 base pairsencoding the prothrombin gene. Probes 10861 (SEQ ID NO:264) and 9828(SEQ ID NO:265) were used to PCR amplify a region of human genomic DNAspanning about 300 base pairs encoding the Factor V gene. Probes PT5 and10861 have phosphorothioate linkages between the first five bases at the5′ end.

The Factor V and Prothrombin fragments were co-amplified in one PCRreaction under the following conditions:   5 μL 10 × PCR buffer   5 μL25 mM MgCl₂   1 μL 10 mM dNTPs   1 μL probe PT5 (50 pmol)   1 μL probePT6 (50 pmol)   1 μL probe 10861 (50 pmol)   1 μL probe 9828 (50 pmol)  1 μL Human genomic DNA (40 ng)   36 μL water 1.25 U Taq

The PCR cycling parameters were as follows: 94° C., 2 minutes; (94° C.,30 seconds; 60° C., 1 minutes; 70° C., 1 minutes)×40; 70° C., 5 minutes.Fifty units of T7 gene 6 Exonuclease (USB Amersham) were added to 25 μLof the PCR reaction and the solution was incubated for 30 minutes at 37°C. Magnetic silica (Promega, A1330) was used to remove free nucleotidesfrom the solution and the remaining DNA was eluted with 100 μL of water.

The Prothrombin interrogation probes used are 11265 (SEQ ID NO:268) thatmatches mutant prothrombin sequence and 11266 (SEQ ID NO:269) thatmatches wild type prothrombin sequence. Each of those probes has adestabilizing mutation eight bases from the 3′ end as discussed inExample 30. The Factor V interrogation probes used are 9919 (SEQ IDNO:270) that matches wild types Factor V sequence and 11432 (SEQ IDNO:267) that matches Factor V Leiden mutation sequence.

Four microliters of the eluted DNA were interrogated with eachinterrogation probe independently and also with the Factor V andProthrombin mutant probes conjointly in one reaction. The interrogationreactions were assembled as follows.  4 μL DNA (PCR product, Exo6treated and purified) 150 pmol each interrogation oligo water added to afinal volume of 20 μL

The reactions were incubated at 95° C. for 3 minutes and then at 37° C.for 10 minutes. Twenty microliters of the standard master mix was thenadded and the reaction incubated at 37° C. for 15 minutes. One hundredmicroliters of the L/L reagent were then added and the light outputmeasured in a Turner® TD20/20 luminometer. The master mix contains thefollowing. 71 μL water 20 μL 10 × DNA pol buffer  5 μL 40 mM NaPPi  2 μL10 μM ADP  1 μL 1 unit/μL NDPK  1 10 unit/μL Klenow exo-

The light output was as follows. Interrogation Genomic Genomic oligo DNA1 DNA 2  9919 (FV wt) 431 424 11432 (FV mut) 45 57 11266 (Pt wt) 902 87811265 (Pt mut) 145 161 11432 + 11265 77 98 no oligo 44 57

These data indicate that the both genomic DNAs are from individuals wildtype for Factor V and for wild type Prothrombin.An additional 96 clinical genomic DNA samples were interrogated asdescribed above. All the data fit into the following equation forcalling the genotype.$\frac{{rlu}\quad{both}\quad{wild}\quad{type}\quad{probes}}{{{rlu}\quad{both}\quad{wild}\quad{type}} + {{rlu}\quad{both}\quad{mutant}\quad{probes}}} > 0.75$

This equation is the analytical output from the interrogation includingboth wild type probes divided by the analytical output from both wildtype probes added to the analytical output from both mutant probes. Ifthat value is greater than 0.75 then the sample is homozygous wild typeat both loci. If that value is less than 0.75 then there is goodlikelihood that at least one allele at least one of the loci is mutantand the sample should be further analyzed for the genotype at each locusseparately. PT5 5′ ATAGCACTGGGAGCATTGAGGC 3′ SEQ ID NO:104 PT6 5′GCACAGACGGCTGTTCTCTT 3′ SEQ ID NO:105 10861 5′ TGCCCAGTGCTTAACAAGACCA 3′SEQ ID NO:264 9828 5′ TGTTATCACACTGGTGCTAA 3′ SEQ ID NO:265 11265 5′GTGATTCTCAGCA 3′ SEQ ID NO:268 11266 5′ GTGATTCTCAGCG 3′ SEQ ID NO:2699919 5′ GACAAAATACCTGTATTCCTCG 3′ SEQ ID NO:270 11432 5′GACAAAATACCTGTATTCCTTG 3′ SEQ ID NO:267

EXAMPLE 72 Shrimp Alkaline Phosphatase as dNTP Removal Reagent

This Example examines pretreatment of the PCR product with shrimpalkaline phosphatase to shorten the protocol using magnetic silica forpurification of PCR products.

The standard protocol for purification of PCR products prior tointerrogation can be broken down into the following steps.

-   1. Twenty five microliters PCR product are added to 175 μL Magnetic    Silica (Promega, A1330)-   2. Three 200 μL washes with 70% ethanol-   3. Dry DNA bound to Magnetic Silica 10 minutes at room temperature-   4. Elute DNA from Magnetic Silica with 50 μL water-   5. Add 150 μL Magnetic Silica to the 50 μL DNA in water-   6. Three 200 μL washes with 70% ethanol-   7. Elute DNA from Magnetic Silica with 100 μL water    The revised protocol is as follows.-   1. Twenty five microliters of PCR product are added to 1 unit of    Shrimp Alkaline Phosphatase-   2. Incubate 15 minutes at room temperature-   3. Add 175 μL Magnetic Silica-   4. Three 200 μL washes with 70% ethanol-   5. Dry DNA bound to Magnetic Silica 10 minutes at room temperature-   6. Elute DNA from Magnetic Silica with 100 μL water

In this example, the standard protocol was compared to the revisedprotocol using Factor V Leiden and wild type PCR product andinterrogation oligonucleotides as described in Example 32. The wild-typeoligonucleotides used were FV1 (SEQ ID NO:25) and FV2 (SEQ ID NO:26).The mutant oligonucleotides used were FV3 (SEQ ID NO:27) and FV4 (SEQ IDNO:28). The probes used were FV7 (wild-type) (SEQ ID NO:110) and FV8(mutant) (SEQ ID NO:111). The interrogation reaction was added to 100 μLof L/L reagent (Promega, F202A)and the relative light units determinedon a Turner® TD20/20 luminometer. The values for each are shown below.Wild Type Mutant No Protocol Interro* Interro Interro Discrim* Standard1867 289.7 165.0 13.7 (2 washes) Standard 2144 775.6 623.6 10.0 (1 wash)Revised 1475 393.4 190.7 6.3*Interro = interrogation;Discrim = discrimination

The Standard Protocol with two sets of washes worked best based onabsolute background and fold discrimination over background.Pre-treating with Shrimp Alkaline Phosphatase before a single round ofDNA capture and washes as in the revised protocol gave relatively lowbackgrounds, but the discrimination ratio was lower. FV1 5′CTAATCTGTAAGAGCAGATCCCTGGACAGGCGAG SEQ ID NO:25GAATACAGAGGGCAGCAGACATCGAAGAGCT 3′ FV2 5′AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTC SEQ ID NO:26GCCTGTCCAGGGATCTGCTCTTACAGATTAGAGCT 3′ FV3 5′CTAATCTGTAAGAGCAGATCCCTGGACAGGCAAG SEQ ID NO:27GAATACAGAGGGCAGCAGACATCGAAGAGCT 3′ FV4 5′AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTT SEQ ID NO:28GCCTGTCCAGGGATCTGCTCTTACAGATTAGAGCT 3′ FV7 5′ GACAAAATACCTGTATTCCTCG 3′SEQ ID NO:110 FV8 5′ GACAAAATACCTGTATTCCTTG 3′ SEQ ID NO:111

EXAMPLE 73 Analysis of SNP Heterozygosity Level in DNA Isolated fromPlant Materials

Eight different rice DNA samples, with varying amounts of two allelesdiffering at an SNP site, were analyzed to determine the ability ofpyrophosphorylation to detect the degree of heterozygosity in a plantsample. The DNA genotypes (G and T) are described in Example 39.

Eight coded heterozygous rice DNA samples and two homozygous rice DNAsamples were obtained (Texas A&M, Crop Biotechnology Center) and PCRamplified with primers RS1 (SEQ ID NO:129) and RS2 (SEQ ID NO:130) asdescribed in Example 39. The resulting PCR products were then treatedwith T7 Exonuclease 6 and purified as described in Example 39. Theresulting DNA was interrogated by combining 4 μL of the PCR product with150 pmoles of interrogation oligonucleotide and water to a final volumeof 20 μL. This solution was incubated at 95° C. for 2 minutes, then at37° C. for 10 minutes. Interrogation oligonucleotides used were RS3 (SEQID NO:131), RS4 (SEQ ID NO:132), and none. Twenty microliters of mastermix were then added and the solution further incubated at 37° C. for 15minutes. These solutions were then combined with 100 μL of L/L reagent(Promega, F202A) and light output measured in a Berthold Eg&G microlumatplus luminometer. The relative light units (rlu) were corrected for nooligonucleotide background and are listed below: G (RS3) T(RS4) SampleAllele Allele % G % T 1 100,366 119,046 45.7 54.3 2 83,428 163,241 33.866.2 3 90,309 90,628 49.9 50.1 4 168,074 35,835 82.4 17.6 5 173,42231,403 84.7 15.3 6 166,516 13,692 92.4 7.6 7 171,933 17,384 90.8 9.2 8103,047 1,724 98.4 1.6

These G:T ratios were further confirmed by the following study. Theeight heterozygote samples and the two homozygote samples werere-amplified as previously described, but the PCR reaction also included1 μL of ³²PdATP and ³²PdCTP. Ten microliters of the resulting PCRproduct were then digested with 1 μL restriction endonuclease AccI in2.5 mM MgCl₂ in the PCR buffer for one hour at 37° C. AccI cuts the Gallele PCR product into a 120 bp doublet, but will not cut the 240 bpfragment from the T allele PCR product. The digest was run on a 10%acrylamide TBE gel, dried for one hour at 65° C. and resulting bandsquantified for one hour on a Molecular Dynamics Fluoroimager screen. Thefollowing values were obtained. Sample % G % T 1 47.6 52.4 2 35.8 64.2 357.2 42.8 4 77.6 22.3 5 81.1 18.9 6 89.2 10.8 7 85.4 14.6 8 nd nd

No values were generated for sample 8 because the PCR reaction was notsuccessful. The correlation coefficient between the two data sets was0.992036. RS1 5′ CCCAACACCTTACAGAAATTAGC 3′ SEQ ID NO:129 RS2 5′TCTCAAGACACAAATAACTGCAG 3′ SEQ ID NO:130 RS3 5′ AGAACATCTGCAAGG 3′ SEQID NO:131 RS4 5′ AGAACATCTGCAAGT 3′ SEQ ID NO:132

EXAMPLE 74 Deoxyadenosine Triphosphate as a Luciferase Substrate

In the usual interrogation assay, the pyrophosphorylation activity of apolymerase is used to break down nucleotides at the 3′ end of ahybridized probe into its dNTP subunits. The terminal phosphate group isthen transferred from the released dNTPs to ADP by NDPK activity,thereby forming ATP. The ATP in turn is used as a substrate byluciferase resulting in light production. In this Example thepyrophosphorylation reaction is performed to determine if the dATPreleased by the pyrophosphorylation of a hybridized interrogation probecan be detected sufficiently to permit for discrimination from amismatched interrogation probe in the absence of NDPK and ADP.

It has been previously shown that dATP is an inefficient substrate forlight production by luciferase relative to ATP. The use of dATP byluciferase produces about 2% of the light output as the use of ATP.However, dATP is used by luciferase much more efficiently than any ofthe other nucleoside triphosphates or deoxynucleoside triphosphates(Analytical Biochemistry 134:187-189, 1983).

The targets in this Example are 10800 (SEQ ID NO:171) and 10801 (SEQ IDNO:172) and represent wild-type cytomegalovirus. The probes in thisexample are wild-type 10337 (SEQ ID NO:284) and mutant 10338 (SEQ IDNO:285). Six replicates of the following solutions were assembled. μL μLμL Water CMV Target CMV Probe Match 18 1, WT* 1, WT Mismatch 18 1, Mut2*1, Mut2 Probe only 19 none 1, WT Target only 19 1, WT none No DNAcontrol 20 none none*WT = wild type; MUT = mutant

These solutions were incubated at 86° C. for 5 minutes, then cooled atroom temperature for 45 minutes. Two different master mix solutions wereassembled. Master Mix 1 Master Mix 2 Water 288 μL 300 μL 10 × DNA Polbuffer  80 μL  80 μL 40 mM NaPPi  10 μL  10 μL Klenow exo-  10 μL  10 μL(1 U/μL) NDPK (1 U/μL)  4 μL none ADP 10 μM  8 μL none

Twenty microliters of each type of master mix were added to three of thesix replicates of the sample type: match, mismatch, probe only, targetonly and no DNA control. The resulting solutions were incubated at 37°C. for 15 minutes, then added to 100 μL of L/L reagent (Promega, F202A)and the light output determined on a Turner® TD20/20 luminometer. Theresults of the study are shown below. Solution Master Mix 1 Master Mix 2Match 1469 1522 1536 1.702 2.081 1.723 Mismatch 126.6 127.2 126.4 1.0811.090 0.988 Probe only 23.7 23.9 27.6 0.681 10.93 3.068 Target only 20.320.8 21.1 0.564 0.640 1.315 No DNA 18.2 23.2 16.5 0.863 0.365 0.378

This Example demonstrates that discrimination can be achieved in theusual interrogation assay that is lacking in NDPK and ADP. The degree ofdiscrimination is much lower than it would be in the presence of NDPKand ADP. Two of the values using probe only solution with master mix twoare artificially high, presumably due to contamination. Thesemeasurements were repeated and the following values obtained when 35 μLof the interrogation reaction were combined with 100 μL of L/L reagentand the light output determined on a Turner® TD20/20 luminometer.Solution Master Mix 2 Match 2.381 2.347 2.402 2.235 3.150 2.800 Mismatch1.331 1.312 1.290 1.308 1.260 1.261 Probe only 1.308 1.289 1.334 1.3131.311 1.315 Target only 1.142 1.233 1.229 1.297 1.190 2.353 No DNA 1.0501.025 1.036 0.977 1.104 0.898

10800 5′ CGTGTATGCCACTTTGATATTACACCCATGAACG SEQ ID NO:171TGCTCATCGACGTGAACCCGCACAACGAGCT 3′ 10801 5′CGTTGTGCGGGTTCACGTCGATGAGCACGTTCAT SEQ ID NO:172GGGTGTAATATCAAAGTGGCATACACGAGCT 3′ 10337 5′ CACTTTGATATTACACCCATG 3′ SEQID NO:284 10338 5′ CACTTTGATATTACACCCGTG 3′ SEQ ID NO:285

EXAMPLE 75 Detection of Human Immunodeficiency Virus (HIV)Drug-Resistant Mutants

Chemotherapeutic selection pressure in vivo often results in mutationswithin the genome of the infectious agent that the drug is intended todestroy. This demonstration of evolutionary adaptation is widelyreported for human immunodeficiency virus (HIV) under the selectivepressure of protease inhibitors or reverse transcriptase inhibitors(Martinez-Picado, J. Virology, 73:3744-3752, 1999; Back, EMBO J.,15:4040-4049, 1996).

The first viral mutants to be selected during therapy are typicallythose with single-amino-acid substitutions. Some of the nucleotides ofthe HIV reverse transcriptase (RT) and protease genes are known in theart to be “hotspots” for developing such point mutations. Additionalmutations accumulate with ongoing therapy. After about 6 months to 1year of treatment with AZT, HIV typically mutates the RT gene and sobecomes resistant to treatment.

The ability to detect and identify such viral mutant genomes in areliable and sensitive assay would assist with understanding theprogression of the infection and with developing the best treatmentregimens for infected individuals. Switching to a different treatmentcourse before or as soon as a resistant mutant virus takes hold isimportant in prolonging patient life.

This Example demonstrates that drug resistant mutations that occurwithin the HIV-1 reverse transcriptase gene, when under the selectivepressure of reverse transcriptase inhibitors, such as the nucleosideanalog drugs AZT and ddI, can be detected using the process of theinvention. Three specific “hotspot” sites of RT mutation were chosen forstudy. These three mutations all exist within a short region of the RTgene, spanning about 10 amino acids, from codon 65 to 75 of the protein.

Codon 67 (Site 1) of RT changes from GAC to AAC in the presence of thedrug AZT, codon 70 (Site 2) changes from AAA to AGA in the presence ofAZT, and codon 75 (Site 3) changes from GTA to ATA in the presence ofthe combination of drugs AZT and ddI. Target oligonucleotides weresynthesized to span codons 65 through 81 of the RT genome of HIV-1strain HXB2 wild type genome as well as oligonucleotides that vary onlyat one position as defined above for Site 1, Site 2, and Site 3 pointmutations. Probe oligonucleotides exactly complementary to the wild typetarget and to the mutant targets at these three sites were alsosynthesized. The sequence and names of these oligonucleotides are listedbelow.

The probe oligonucleotides were dissolved in TE Buffer to a finalconcentration of 0.5 μg/μL. The target oligonucleotides were dissolvedin TE Buffer to a final concentration of 5 μg/mL. One microliter oftarget was combined with 1 μL of probe and 18 μL of water; and for thecontrols, 1 μL of each oligonucleotide was combined with 19 μL of water.These solutions were then heated at 95° C. for 3 minutes and cooled atroom temperature for 10 minutes. Twenty microliters of master mix werethen added to each tube. The master mix is described below. Master Mix:10 × DNA Polymerase buffer (Promega, M195A) 120 μL 40 mM Sodiumpyrophosphate  15 μL Klenow exo- enzyme (1 U/μL; Promega, M218A)  15 μLNDPK (1 U/μL)  6 μL ADP (10 μM)  12 μL Nanopure water 432 μL

The tubes with the master mix added were then incubated for 15 minutesat 37° C. Five microliters of the solutions were then combined with L/Lreagent (Promega, F202A) and the light output was measured on a Turner®TD20/20 luminometer. The relative light unit (rlu) data obtained arelisted below. Solution Target Probe Reading 1 Reading 2 Reading 3 1)11814 — 2.55 3.82 10.78 (wt*) 2) 11815 — 2.54 2.57 2.99 (mut1*) 3) —11808 162.8 207.2 165.5 (wt1) 4) — 11809 2.81 2.17 2.20 (mut1) 5) 11816— 3.84 3.98 3.81 (mut2) 6) — 11810 4.57 4.77 5.29 (wt2) 7) — 11811 3.843.98 3.81 (mut2) 8) 11817 — 2.04 1.64 1.44 (mut3) 9) — 11812 2.36 2.572.41 (wt3) 10) — 11813 4.05 2.06 1.77 (mut3) 11) 11814 11808 418.7 711.6682.1 (wt) (wt1) 12) 11814 11809 20.69 29.05 21.25 (wt) (mut1) 13) 1181511808 218.4 185.6 118.1 (mut1) (wt1) 14) 11815 11809 682.6 737.8 599.7(mut1) (mut1) 15) 11814 11810 1055.0 920.2 744.7 (wt) (wt2) 16) 1181411811 175.3 188.1 171.1 (wt) (mut2) 17) 11815 11810 136.9 121.0 114.4(mut2) (wt2) 18) 11815 11811 822.3 865.9 729.0 (mut2) (mut2) 19) 1181411812 31.49 33.22 43.83 (wt) (wt3) 20) 11814 11813 2.55 3.79 2.49 (wt)(mut3) 21) 11815 11812 5.26 6.00 6.33 (mut2) (wt3) 22) 11815 11813 77.5878.46 82.85 (mut2) (mut3) 23) no DNA 2.18 2.48 1.37*wt = wild type; mut = mutant. wt and mut a, 2, and 3 are definedhereinafter.

All three HIV RT drug-resistance mutations were detectable withdiscrimination of mutant:wild type rlu ratios ranging from about 3 toabout 7. Probe 11808, which is directed to site one and is completelycomplementary to wild type target, had high background values whentested alone. The other oligonucleotides all had acceptably low levelsof background.

Target and Probe Sequences 11808 5′ CCATTTAGTACTGTCT 3′ SEQ ID NO:271HIV WT Probe Site 1 11809 5′ CCATTTAGTACTGTTT 3′ SEQ ID NO:272 HIVMutant Probe Site 1 11810 5′ CTAGTTTTCTCCATTT 3′ SEQ ID NO:273 HIV WTProbe Site 2 11811 5′ CTAGTTTTCTCCATCT 3′ SEQ ID NO:274 HIV Mutant ProbeSite 2 11812 5′ TTCTCTGAAATCTACT 3′ SEQ ID NO:275 HIV WT Probe Site 311813 5′ TTCTCTGAAATCTATT 3′ SEQ ID NO:276 HIV Mutant Probe Site 3 118145′ AAAAAAGACAGTACTAAATGGAGAAAACTAGTA SEQ ID NO:277 GATTTCAGAGAACTTAA 3′HIV WT Target 11815 5′ AAAAAAAACAGTACTAAATGGAGAAAACTAGTAG SEQ ID NO:278ATTTCAGAGAACTTAA 3′ HIV Mutant Target Site 1 11816AAAAAAGACAGTACTAGATGGAGAAAACTAGTAGATT SEQ ID NO:279 TCAGAGAACTTAA 3′ HIVMutant Target Site 25′ 11817 5′ AAAAAAGACAGTACTAAATGGAGAAAACTAA SEQ IDNO:280 TAGATTTCAGAGAACTTAA 3′ HIV Mutant Target Site 3

EXAMPLE 76 Interrogation Assay at 70° C. Using Pfu NDPK and Tvu DNAPolymerase

In this Example, a mutation that exists in the prohibitin gene in thepopulation was interrogated using synthetic templates and the one-stepinterrogation assay. The assay was performed with two thermostableenzymes: NDPK of Pyrococcus furiosis (Pfu) and the DNA Polymerase ofThermoactinomyces vulgaris (Tvu).

One microliter of each wild type target oligonucleotide 9354 (SEQ IDNO:4) and 9355 (SEQ ID NO:5), at a concentration of 2.5 μg/mL in water,were combined with 1 μL of either 9695 wild type probe (SEQ ID NO:281)or 9498 mutant probe (SEQ ID NO:282)(1 mg/ml) and 18 μL water.Similarly, one microliter of mutant target oligonucleotides 9356 (SEQ IDNO:6) and 9357 (SEQ ID NO:7) were combined with 1 μL of either probe9695 or probe 9498 oligonucleotides and 18 μL water. For the controlsolutions, 1 μL of each oligonucleotide was combined with 19 μL of waterin separate tubes. These solutions were then heated to 95° C. for 5minutes and cooled for 10 minutes at room temperature.

The following master mix was assembled. 10 × DNA Polymerase Buffer(Promega, M195A) 100 μL 25 mM MgCl₂ 100 μL 40 mM Sodium Pyrophosphate 25 μL 1 μM ADP  10 μL Tvu DNA Polymerase (5 U/μL)  5 μL Pfu NDPK (0.5U/μL)  5 μL Nanopure water 255 μL

Twenty microliters of master mix were added to each solution and theywere then incubated at 70° C. for 10 minutes. Four microliters of eachreaction were then combined with 100 μL of L/L reagent (Promega, F202A)and the luminescence (rlu) was measured on a TMDE#14728 luminometer. Theresults are listed below. Solution Target Probe rlu net rlu 1. — — 9.9392. wild type — 18.88 3. mutant — 28.57 4. — wild type 10.10 5. — mutant9.67 6. wild type wild type 583.9* 564.8 7. wild type mutant 24.1* 5.48. mutant wild type 40.7* 11.9 9. mutant mutant 840.6* 812.2*Average of duplicate measurements

This combination of enzymes provides excellent discrimination betweenwild type and mutant prohibitin sites.

Probe and Target Sequences 9354 5′ CTGCTGGGGCTGAACATGCCTGCCAAAGACGTGTSEQ ID NO:4 CCGACCTACGTTCCTGGCCCCCTCGAGCT 3′ Prohibitin wild typestrand - target 9355 5′ CGAGGGGGCCAGGAACGTAGGTCGGACACGTCTT SEQ ID NO:5TGGCAGGCATGTTCAGCCCCAGCAGAGCT 3′ Prohibitin wild type strand - target9356 5′ CTGCTGGGGCTGAACATGCCTGCCAAAGATGTGT SEQ ID NO:6CCGACCTACGTTCCTGGCCCCCTCGAGCT 3′ Prohibitin mutant strand - probe 93575′ CGAGGGGGCCAGGAACGTAGGTCGGACACATCTT SEQ ID NO:7TGGCAGGCATGTTCAGCCCCAGCAGAGCT 3′ Prohibitin mutant strand - probe 96955′ CTGAACATGCCTGCCAAAGACG 3′ SEQ ID NO:281 Prohibitin wild type probe9498 ′ CTGAACATGCCTGCCAAAGATG 3′ SEQ ID NO:282 Prohibitin mutant probe

EXAMPLE 77 Interrogation for Factor V Leiden Mutation: Mass SpectroscopyAnalysis

This Example demonstrates that nucleotides released from the 3′-terminusof a hybridized probe by a process of the invention can be detected bymass spectroscopy. Probes PT5 (SEQ ID NO:104) and PT6 (SEQ ID NO:105)are used to PCR-amplify a region of human genomic DNA spanning about 500base pairs encoding the prothrombin gene. Probe PT5 has phosphorothioatelinkages between the first five bases at the 5′ end. The PCR reactionconditions are detailed in Example 71. The PCR product is treated withT7 gene 6 Exonuclease (USB Amersham) and separated from free nucleotidesas described in Example 71.

The prothrombin interrogation probes are 11265 (SEQ ID NO:268), that istotally complementary to a segment of the mutant prothrombin sequence,and 11266 (SEQ ID NO:269), that is totally complementary to a segment ofthe wild-type prothrombin sequence. Each of these probes has adestabilizing mutation eight bases from the 3′-end as discussed inExample 30.

The purified PCR product is interrogated with each interrogation probe.Two separate interrogation reactions for each of the interrogationprobes are assembled as follows.  40 μL PCR product 1.5 nmolInterrogation oligoWater is added to a final volume of 50 μL.

The reactions are incubated at 95° C. for 3 minutes and then at 37° C.for 15 minutes for Klenow exo− reactions.

A replicate set of solutions is incubated at 95° C. for 3 minutes andthen 55° C. for 15 minutes for the Tne triple mutant reactions.

Fifty microliters of the appropriate master mix are then added. Onemaster mix contains Klenow exo− polymerase and yeast NDPK. The othermaster mix contains Tne triple mutant polymerase and Pfu NDPK. Thecompositions of the master mixes are as described in Example 57. Thereaction containing Klenow exo− proceeds at 37° C. for 15 minutes. Thereaction containing Tne triple mutant polymerase proceeds at 55° C. for15 minutes.

The presence or absence of released nucleotides, converted to ATP, isanalyzed for by silicon desorption ionization mass spectroscopy (Wei, J.et al. Nature. 399:243-246, 1999). This method is sensitive to femtomoleand attomole levels of analyte. The samples are prepared as described inthat paper. An observance of released nucleotide from either of thereactions containing the mutant probe, 11265, at levels greater thanbackground, indicates the presence of a mutant prothrombin gene in thegenomic DNA sample assayed. An observance of released nucleotide fromthe either reaction containing the wild-type probe, 11266, at levelsgreater than background, indicates the presence of a wild-typeprothrombin gene in the genomic DNA sample assayed. PT5 5′ATAGCACTGGGAGCATTGAGGC 3′ SEQ ID NO:104 PT6 5′ GCACAGACGGCTGTTCTCTT 3′SEQ ID NO:105 11265 5′ GTGATTCTCAGCA 3′ SEQ ID NO:268 11266 5′GTGATTCTCAGCG 3′ SEQ ID NO:269

EXAMPLE 78 Multiplex Interrogation for Factor V Leiden and ProthrombinMutation: Mass Spectroscopy Analysis

This Example demonstrates that nucleotides released from the 3′-terminusof a hybridized probe in a multiplex reaction by a process of theinvention can be detected by mass spectroscopy and thereby determinewhether a mutant allele exists at one of the loci being studied.

Probes PT5 (SEQ ID NO:104) and PT6 (SEQ ID NO:105) are used toPCR-amplify a region of human genomic DNA spanning about 500 base pairsencoding the prothrombin gene. Probes 10861 (SEQ ID NO:264) and 9828(SEQ ID NO:265) are used to PCR amplify a region of human genomic DNAspanning about 300 base pairs encoding the Factor V gene. These probesand the PCR reaction conditions are detailed in Example 71. Probes PT5and 10861 have phosphorothioate linkages between the first five bases atthe 5′ end. The PCR product is treated with T7 gene 6 Exonuclease (USBAmersham) and separated from free nucleotides as described in Example71.

The prothrombin interrogation probe, 11265 (SEQ ID NO:268), is totallycomplementary to a segment of the mutant prothrombin sequence. TheFactor V interrogation probe, 11432 (SEQ ID NO:267), is totallycomplementary to a segment of the mutant Factor V Leiden mutationsequence. Each of these probes has a destabilizing mutation eight basesfrom the 3′-end as discussed in Example 30.

The PCR products are synthesized, Exo 6 treated, and purified asdescribed in Example 71. The interrogation reactions are assembled with40 μL of each PCR product and 1.5 nmol of each interrogation probe.Water is added to a final volume of 100 μL. These reactions areassembled in duplicate so that one can be assayed with Klenow exo−polymerase and yeast NDPK at 37° C., while the other is assayed with Tnetriple mutant polymerase and Pfu NDPK at 70° C.

These assembled reactions are incubated at 95° C. for 3 minutes and thenat 37° C. for 10 minutes. The assembled reactions may be lyophilized todecrease the volume. The two different master mixes are assembled asdescribed in Example 77. An equal volume of each master mix isseparately added to the reaction solutions described above. Then thesolution containing Klenow exo− as the polymerase is incubated at 37° C.for 15 minutes, while the solution containing Tne triple mutantpolymerase is incubated at between 55° C. and 70° C.

The presence or absence of released nucleotides, converted to ATP, isanalyzed for by silicon desorption ionization mass spectroscopy (Wei, J.et al. Nature. 399:243-246, 1999). This method is sensitive to femtomoleand attomole levels of analyte. The samples are prepared as described inthat paper. Essentially, analytes are dissolved in a deionizedwater/methanol mixture (1:1) at concentrations typically ranging from0.001 to 10.0 μM. Aliquots (at least 0.5 to 1.0 μL, corresponding to atleast 0.5 femtomol to 100 picomol analyte) of solution are depositedonto the porous surfaces and allowed to dry before mass spectrometryanalysis. These experiments are performed on a Voyager DE-STR,time-of-flight mass spectrometer (PerSeptive Biosystems) using a pulsednitrogen laser (Laser Science) operated at 337 nm. Once formed, ions areaccelerated into the time-of-flight mass analyzer with a voltage of 20kV. Other liquid chromatography-mass spectrometry (LC-MS)instrumentation may also be used for analysis (Niessen W. J. ChromatograA 794: (407-435, 1998).

An observance of released nucleotide from either of the reactionscontaining the two mutant probe, at levels greater than background,indicates the presence of a at least one mutant prothrombin or Factor VLeiden allele in the genomic DNA sample assayed. 10861 5′TGCCCAGTGCTTAACAAGACCA 3′ SEQ ID NO:264 9828 5′ TGTTATCACACTGGTGCTAA 3′SEQ ID NO:265 PT5 5′ ATAGCACTGGGAGCATTGAGGC 3′ SEQ ID NO:104 PT6 5′GCACAGACGGCTGTTCTCTT 3′ SEQ ID NO:105 11265 5′ GTGATTCTCAGCA 3′ SEQ IDNO:268 11432 5′ GACAAAATACCTGTATTCCTTG 3′ SEQ ID NO:267

EXAMPLE 79 Interrogation Using Fluorescence

This Example demonstrates that nucleotides released from the 3′-terminusof a probe hybridized to a target nucleic acid of interest by a processof the invention can be detected by mass spectrometry or by fluorimetricHPLC and thereby provide evidence of the presence or absence of thetarget nucleic acid in a nucleic acid sample or of a specific base at aninterrogation position of the target.

The interrogation probe is designed to have a fluorescent label attachedto the 3′-terminal nucleotide in a manner such that the label does notinterfere with the ability of the depolymerizing enzyme to remove thenucleotide from the probe. Such fluorescent tags, such as fluorescein orrhodamine, can be incorporated into the probe during synthesis with afluorescent molecule attached to the phosphoramadite nucleotide with alinker of at least 6 carbons (Glen Research). Additionally, in thisExample an identical, but unlabeled, probe is used and releasednucleotides are fluorescently labeled only after the nucleotide isreleased from the probe by a process of the invention.

Probes PT5 (SEQ ID NO:104) and PT6 (SEQ ID NO:105) are used toPCR-amplify a region of human genomic DNA spanning about 500 base pairsencoding the prothrombin gene. Probe PT5 has phosphorothioate linkagesbetween the first five bases at the 5′ end. The PCR reaction conditionsare detailed in Example 71. The PCR product is treated with T7 gene 6Exonuclease (USB Amersham) and separated from free nucleotides asdescribed in Example 71.

The prothrombin interrogation probes are 11265 (SEQ ID NO:268), that istotally complementary to a segment of the mutant prothrombin sequence,and 11266 (SEQ ID NO:269), that is totally complementary to a segment ofthe wild-type prothrombin sequence. Each of these probes has adestabilizing mutation eight bases from the 3′ end as discussed inExample 30. Also, each of these probes is synthesized in two forms: withand without a fluorescent nucleotide analog (fluorescein-derivative) atthe 3′-terminal nucleotide position. When the probe has the fluorescenttag, it is incorporated during synthesis of the probe as describedabove.

The purified PCR product is interrogated in separate reactions with eachof the four interrogation probes (wild-type and mutant, with and withoutfluorescent tag). Interrogation reactions for each of the interrogationprobes are assembled as follows.  40 μL PCR product 1.5 nmolInterrogation oligoWater is added to a final volume of 50 μL.

The reactions are incubated at 95° C. for 3 minutes and then at 37° C.for 15 minutes.

Fifty microliters of master mix are then added. The composition of themaster mix containing Klenow exo− is described in Example 57 with theexception that there is no ADP and no NDPK. The reaction then proceedsat 37° C. for 15 minutes. The two reactions that do not containfluorescent-labeled nucleotides are further treated to label thereleased nucleotides with a fluorescein tag (Jain, R. et al. BiochemBiophys Res Commun. 200:1239-1244, 1994; Shuker, D. et al. IARC Sci Publ124:227-232, 1993).

The solutions are then split in half and analyzed using two differentmethods. In one method, the presence or absence of released nucleotidesin the solutions is analyzed by silicon desorption ionization massspectroscopy (Wei, J. et al. Nature. 399:243-246, 1999). This method issensitive to femtomole and attomole levels of analyte. The samples areprepared for spectrometry as described in that paper. Essentially,analytes are dissolved in a deionized water/methanol mixture (1:1) atconcentrations typically ranging from 0.001 to 10.0 μM. Aliquots (atleast 0.5 to 1.0 μL, corresponding to at least 0.5 femtomol to 100picomol analyte) of solution are deposited onto the porous surfaces andallowed to dry before mass spectrometry analysis. These experiments areperformed on a Voyager DE-STR, time-of-flight mass spectrometer(PerSeptive Biosystems) using a pulsed nitrogen laser (Laser Science)operated at 337 nm. Once formed, ions are accelerated into thetime-of-flight mass analyzer with a voltage of 20 kV. Other liquidchromatography-mass spectrometry (LC-MS) instrumentation may also beused for analysis (Niessen W. J. Chromatogra A 794: (407-435, 1998).

In a second method, the presence or absence of released nucleotides inthe solutions is analyzed by HPLC using a fluorescence detector asdescribed in Jain, et al. Biochem Biophys Res Commun 200:1239-1244, 1994or Levitt, B. et al. Anal Biochem 137:93-100, 1984.

An observance of released nucleotide from either of the reactionscontaining the mutant probe, 11265, at levels greater than background(control reactions that contain no enzyme), indicates the presence of atleast one mutant prothrombin allele in the genomic DNA sample assayed.An observance of released nucleotide from either reaction containing thewild-type probe, 11266, at levels greater than background, indicates thepresence of at least one wild-type prothrombin allele in the genomic DNAsample assayed. PT5 5′ ATAGCACTGGGAGCATTGAGGC 3′ SEQ ID NO:104 PT6 5′GCACAGACGGCTGTTCTCTT 3′ SEQ ID NO:105 11265 5′ GTGATTCTCAGCA 3′ SEQ IDNO:268 11266 5′ GTGATTCTCAGCG 3′ SEQ ID NO:269

EXAMPLE 80 Multiplex Interrogation Using Fluorescent Labels

This example demonstrates that nucleotides released from the 3′-terminusof multiple probes, each hybridized to a target nucleic acid ofinterest, by a process of the invention can be detected by massspectrometry or by fluorimetric HPLC and thereby provide evidence of thepresence or absence of the target nucleic acid in a nucleic acid sampleor of a specific base at an interrogation position of the target.

Each interrogation probe is designed to have a different fluorescentlabel attached to the 3′-terminal nucleotide in a manner such that thelabel does not interfere with the ability of the depolymerizing enzymeto remove the nucleotide from the probe. Such fluorescent tags, such asfluorescein or rhodamine, can be incorporated into the probe duringsynthesis with a fluorescent molecule attached to the phosphoramaditenucleotide with a linker of at least 6 carbons (Glen Research).

Probes PT5 (SEQ ID NO:104) and PT6 (SEQ ID NO:105) are used toPCR-amplify a region of human genomic DNA spanning about 500 base pairsencoding the prothrombin gene. Probes 10861 (SEQ ID NO:264) and 9828(SEQ ID NO:265) are used to PCR amplify a region of human genomic DNAspannning about 300 base pairs encoding the Factor V gene. These probesand the PCR reaction conditions are detailed in Example 71. The PCRproducts are treated with T7 gene 6 Exonuclease (USB Amersham) andseparated from free nucleotides as described in Example 71.

The prothrombin interrogation probes are 11265 (SEQ ID NO:268), that istotally complementary to a segment of the mutant prothrombin sequence,and 11266 (SEQ ID NO:269), that is totally complementary to a segment ofthe wild-type prothrombin sequence. Each of these probes has adestabilizing mutation eight bases from the 3′-end as discussed inExample 30. Also, each of these probes is synthesized with a fluorescentnucleotide analog at the 3′-terminal nucleotide position. Theprothrombin probes are tagged with fluorescein; the factor V probes aretagged with rhodamine.

The purified PCR products are interrogated in separate reactions witheither both wild-type probes or both mutant probes. Interrogationreactions are assembled as follows:  40 μL each of the two PCR products1.5 nmol each of the wild type or each of the mutant labeledinterrogation oligoswater is added to a final volume of 100 μL.

The reactions are incubated at 95° C. for 3 minutes and then at 37° C.for 15 minutes. The reactions are then lyophilized to a final volume of20 μL.

Twenty microliters of master mix are then added. The composition of themaster mix containing Klenow exo− is described in Example 57 with theexception that there is no ADP and no NDPK. The reaction then proceedsat 37° C. for 15 minutes.

The solutions are then split in half and analyzed using two differentmethods. In one method, the presence or absence of released nucleotidesin the solutions is analyzed by silicon desorption ionization massspectroscopy (Wei, J. et al. Nature. 399:243-246, 1999). This method issensitive to femtomole and attomole levels of analyte. The samples areprepared for spectrometry as described in that paper. Essentially,analytes are dissolved in a deionized water/methanol mixture (1:1) atconcentrations typically ranging from 0.001 to 10.0 μM. Aliquots (atleast 0.5 to 1.0 μL, corresponding to at least 0.5 femtomol to 100picomol analyte) of solution are deposited onto the porous surfaces andpermitted to dry before mass spectrometry analysis.

These studies are performed on a Voyager DE-STR, time-of-flight massspectrometer (PerSeptive Biosystems) using a pulsed nitrogen laser(Laser Science) operated at 337 nm. Once formed, ions are acceleratedinto the time-of-flight mass analyser with a voltage of 20 kV. Otherliquid chromatography-mass spectrometry (LC-MS) instrumentation may beused for analysis (Niessen W. J. Chromatogra A 794:407-435, 1998)

In a second method, the presence or absence of released nucleotides inthe solutions is analyzed by HPLC using a fluorescence detector asdescribed in Jain, et al. Biochem Biophys Res Commun 200:1239-1244, 1994or Levitt, B. et al. Anal Biochem 137:93-100, 1984.

An observance of released nucleotide from the reactions containing themutant probes, at levels greater than background (control reactions thatcontain no enzyme), is indicative of the presence of at least one mutantprothrombin or Factor V Leiden allele in the genomic DNA sample assayed.An observance of released nucleotide from the reaction containing thewild-type probes, at levels greater than background, is indicative ofthe presence of at least one wild-type prothrombin or Factor V allele inthe genomic DNA sample assayed. PT5 5′ ATAGCACTGGGAGCATTGAGGC 3′ SEQ IDNO:104 PT6 5′ GCACAGACGGCTGTTCTCTT 3′ SEQ ID NO:105 10861 5′TGCCCAGTGCTTAACAAGACCA 3′ SEQ ID NO:264 9828 5′ TGTTATCACACTGGTGCTAA 3′SEQ ID NO:265

EXAMPLE 81 Exonuclease

In this Example, an interrogation for the presence or absence of aFactor V Leiden mutant allele in a genomic DNA sample is demonstratedwith the use of E. coli Exonuclease III and, in a separate reaction,with the use of E. coli Klenow Fragment.

Probes PT5 (SEQ ID NO:104) and PT6 (SEQ ID NO:105) are used toPCR-amplify a region of human genomic DNA spanning about 500 base pairsencoding the prothrombin gene. Probe PT5 has phosphorothioate linkagesbetween the first five bases at the 5′-end. The PCR reaction conditionsare detailed in Example 71. The PCR product is treated with T7 gene 6Exonuclease (USB Amersham) and separated from free nucleotides asdescribed in Example 71.

The prothrombin interrogation probes are 11265 (SEQ ID NO:268), that istotally complementary to a segment of the mutant prothrombin sequence,and 11266 (SEQ ID NO:269), that is totally complementary to a segment ofthe wild-type prothrombin sequence. Each of these probes has adestabilizing mutation eight bases from the 3′-end as discussed inExample 30.

The purified PCR product is interrogated with each interrogation probe,11265 and 11266. Two separate interrogation reactions are assembled asfollows:  4 μL PCR product 150 pmol interrogation oligowater is added to a final volume of 20 μL.

The reactions are incubated at 95° C. for 3 minutes and then at 37° C.for 15 minutes. Twenty microliters of each of the two master mixes beloware then added to a tube containing the mutant interrogationoligonucleotide and to a tube containing the wild type interrogationoligonucleotide. The reaction proceeds at 37° C. for 15 minutes. Thecomposition of each master mix is: Master mix 1 Master mix 2 10 U Klenow 1 μL 10 U Exonuclease III  1 μL (Promega, M220A) (Promega, M181A) 10 ×enzyme buffer 10 μL 10 × enzyme buffer 10 μL 40 mM NaPPi  5 μL 40 mMNaPPi  5 μL water 84 μl water 84 μlThe released NMPs (reaction 3) are converted to the corresponding NTP bythe enzyme PRPP synthetase as described in Example 86, hereinafter.

One hundred microliters of the L/L Reagent (Promega, F202A) are added,and the light output measured in a luminometer. The presence or absenceof released nucleotides can also be assayed for by silicon desorptionionization mass spectroscopy (Wei, J. et al. Nature. 399:243-246, 1999)as described in Example 80.

An observance of released nucleotide resulting from the Klenow reactioncontaining the mutant probe, indicates the presence of at least onemutant prothrombin allele in the genomic DNA sample assayed. Anobservance of released nucleotide resulting from the Exo III reactioncontaining the mutant probe indicates the presence of at least one wildtype prothrombin allele in the genomic DNA sample assayed. Conversely,an observance of released nucleotide resulting from the Klenow reactioncontaining the wild-type probe, indicates the presence of at least onewild-type prothrombin allele in the genomic DNA sample assayed. Anobservance of released nucleotide resulting from the Exo III reactioncontaining the mutant probe indicates the presence of at least onemutant prothrombin allele. PT5 5′ ATAGCACTGGGAGCATTGAGGC 3′ SEQ IDNO:104 PT6 5′ GCACAGACGGCTGTTCTCTT 3′ SEQ ID NO:105 11265 5′GTGATTCTCAGCA 3′ SEQ ID NO:268 11266 5′ GTGATTCTCAGCG 3′ SEQ ID NO:269

EXAMPLE 82 Interrogation using Fluorescence-II

This example demonstrates that nucleotides released from the 3′-terminusof a probe hybridized to a target nucleic acid of interest by a processof the invention can be detected by mass spectrometry or by fluorimetricHPLC and thereby provide evidence of the presence or absence of thetarget nucleic acid in a nucleic acid sample or of a specific base at aninterrogation position of the target.

The interrogation probe is designed to have a fluorescent label attachedto the 5′-terminal nucleotide. Fluorescent tags, such as fluorescein orrhodamine, can be incorporated into the probe during synthesis with afluorescent molecule attached to the phosphoramadite nucleotide presentat the 5′-end of the oligonucleotide that will be used as a probe (GlenResearch).

Probes PT5 (SEQ ID NO:104) and PT6 (SEQ ID NO:105) are used toPCR-amplify a region of human genomic DNA spanning about 500 base pairsencoding the prothrombin gene. Probe PT5 has phosphorothioate linkagesbetween the first five bases at the 5′ end The PCR reaction conditionsare detailed in Example 71. The PCR product is treated with T7 gene 6Exonuclease (USB Amersham) and purified from free nucleotides asdescribed in Example 71.

The prothrombin interrogation probes are 11265 (SEQ ID NO:268), that istotally complementary to a segment of the mutant prothrombin sequence,and 11266 (SEQ ID NO:269), that is totally complementary to a segment ofthe wild-type prothrombin sequence. Each of these probes has adestabilizing mutation eight bases from the 3′ end as discussed inExample 30. And each of these probes has a label at its 5′-end,incorporated during synthesis of the probe as described above.

The purified PCR product is interrogated in separate reactions with eachof the two interrogation probes (wild-type and mutant). Interrogationreactions, with the target molecule in molar excess over the probemolecule, for each of the interrogation probes are assembled as follows:40 μL PCR product 15 pmol Interrogation oligowater is added to a final volume of 50 μL.

The reactions are incubated at 95° C. for 3 minutes and then at 37° C.for 15 minutes.

Fifty microliters of master mix are then added. The composition of themaster mix containing Klenow exo− is described in Example 57 with theexception that there is no ADP and no NDPK. The reaction then proceedsat 37° C. for 15 minutes. The hybrid is then denatured by incubating thereaction at 95° C. for 3 minutes, adding 100 μL water to dilute theseparated strands and placing the resulting denatured solution tube onice.

The solutions are then split in half and analyzed using two differentmethods. In one method, the size of the labeled probe in the solutionsis analyzed by silicon desorption ionization mass spectroscopy (Wei, J.et al. Nature. 399:243-246, 1999). This method is sensitive to femtomoleand attomole levels of analyte. The samples are prepared forspectrometry as described in that paper. Essentially, analytes aredissolved in a deionized water/methanol mixture (1:1) at concentrationstypically ranging from 0.001 to 10.0 μM. Aliquots (at least 0.5 to 1.0μL, corresponding to at least 0.5 femtomol to 100 picomol analyte) ofsolution are deposited onto the porous surfaces and allowed to drybefore mass spectrometry analysis.

These studies are performed on a Voyager DE-STR, time-of-flight massspectrometer (PerSeptive Biosystems) using a pulsed nitrogen laser(Laser Science) operated at 337 nm. Once formed, ions are acceleratedinto the time-of-flight mass analyzer with a voltage of 20 kV. Otherliquid chromatography-mass spectrometry (LC-MS) instrumentation may alsobe used for analysis (Niessen W. J. Chromatog. A 794:407-435 (1998)

In a second method, the size of the denatured labeled probe strand inthe solution is analyzed by HPLC using a fluorescence detector asdescribed in Jain, et al. Biochem. Biophys. Res. Commun. 200:1239-1244(1994) or Levitt, B. et al. Anal. Biochem. 137:93-100 (1984). The sizeof the denatured labeled probe strand is confirmed on an ABI 377.

The size of the labeled probe strand present in the denatured solutionindicates whether or not a nucleotide was released from the 3′-terminusof the probe, and therefore whether a match or mismatch base pairexisted at the 3′ terminus of the probe/template hybrid. For thedenatured solution containing wild-type probe, the observance of alabeled probe that is shorter than the length of the original probeindicates that there is a matched base at the 3′-terminus of at leastone allele in the original sample and therefore, that at least oneallele in the original sample is wild-type. For the denatured solutioncontaining mutant probe, the observance of a labeled probe that isshorter than the length of the original probe indicates that there is amatched base at the 3′-terminus of at least one allele in the originalsample and therefore, that at least one allele in the original sample iswild-type. In both cases, the analytical output can be quantified todetermine whether the genotype is homozygous or heterozygous at thatlocus. PT5 5′ ATAGCACTGGGAGCATTGAGGC 3′ SEQ ID NO:104 PT6 5′GCACAGACGGCTGTTCTCTT 3′ SEQ ID NO:105 11265 5′ GTGATTCTCAGCA 3′ SEQ IDNO:268 11266 5′ GTGATTCTCAGCG 3′ SEQ ID NO:269

EXAMPLE 83 Detection of E. coli Repetitive Sequence without Nucleic AcidAmplification

In this Example repetitive sequence in E. coli is detected without theneed for amplification of the target sequence prior topyrophosphorylation. This target sequence is denoted as ‘colirep’.

Oligonucleotide 11707 (SEQ ID NO:283) is totally complementary to asegment of colirep DNA sequence. Twelve microliters of oligonucleotide11707 solution (1 mg/mL) were combined with 204 μL of water to makesolution A. Another solution was prepared by combining 4 μL of 11707 (1mg/mL) with 204 μL water and 8 μL 10 mM Tris, pH 8.0 to make solution B.The E. coli is Sigma cat#D4889, E. coli Strain B ultra pure.

Four nanograms (2 μL) E. coli DNA were combined with 18 μL solution Aand with 18 μL solution B in separate tubes. Similarly, 40 ng E. ColiDNA was combined with 18 μL solution A and with 18 μL solution B inseparate tubes. These solutions were then incubated at 92° C. for 3minutes and cooled at room temperature for 15 minutes. The followingmaster mix was assembled: 10 × DNA Polymerase buffer 240 μL 40 mM NaPPi 30 μL Klenow exo- (10 U/μL)  30 μL NDPK (1 U/μL)  12 μL 10 μM ADP(Sigma)  24 μL water 864 μL

Twenty microliters of master mix were added to each reaction and theywere then incubated at 37° C. for 15 minutes. One hundred microliters ofL/L Reagent were then added and the relative light output (rlu)immediately measured with a Turner® TD 20/20 luminometer. The rlu were:Solution rlu-1 rlu-2 rlu-3 Average Tris 2.85 3.562 3.059 3.157 11707 (A)13.69 12.13 13.67 13.16 11707 (B) 7.473 7.234 6.981 7.259 40 ng DNA +Tris 75.62 75.52 73.24 74.79 40 ng DNA + 11707 97.71 134.2 105.1 112.3(A) 40 ng DNA + 11707 81.46 87.56 76.28 81.77 (B)  4 ng DNA + Tris 6.7198.084 5.882 6.895  4 ng DNA + 11707 24.50 25.97 25.17 25.21 (A)  4 ngDNA + 11707 15.69 17.22 16.99 16.63 (B)

The data reflect that oligonucleotide probe 11707 can detect E. coli DNAwithout amplification by a process of the invention.

Interrogation oligonucleotide: 11707 5′ AGTGACTGGGG 3′ SEQ ID NO:283

EXAMPLE 84 PRPP Synthetase, Reactions with Deoxyadenosine Monophosphate

Some schemes for the detection of DNA require the conversion of dAMP,generated by nuclease digestion of DNA, to dATP. This exampledemonstrates that the enzyme PRPP synthetase can perform thetransformation of dAMP to dATP using PRPP as a co-substrate. Inaddition, this transformation can be monitored by luciferase detectionat much higher sensitivities if the dATP formed is used to transform ADPto ATP through the action of NDPK added to the reaction.

The reactions were assembled in duplicate. The concentrations of thereaction components were as follows: 2.9×10⁻⁴ M DAMP in 10 mM Tris pH7.3; 2.9×10⁻⁴ M AMP in 10 mM Tris pH 7.3; 2.6×10⁻⁴ M PRPP in 10 mM TrispH 7.3; 100× dilution of PRPP Syn (PRPP synthetase) (Sigma #P0287) stockenzyme which is at 0.03 U/μL. The components were added to twentymicroliters of PRPP Synthetase Buffer (50 mM triethanolamine, 50 mMpotassium phosphate, pH 7, 0.37 mM EDTA, 10 mM MgCl₂, 1 mg/mL BSA).After incubating for 47 minutes at 37° C., 100 μL LAR Buffer were addedto all reactions along with 10 ng luciferase and the light output of thereactions was immediately measured. The data are presented in RelativeLight Unit table below. PRPP was able to utilize dAMP as a substrate(comparing reaction 1 to 2, 3, 4 and 5). However, the amount of lightproduced by reaction was low, probably due to the fact that luciferaseuses dATP at a much lower efficiency than ATP. Reaction dAMP PRPP PRPPSyn 1 2 μL 2 μL 2 μL 2 2 μL — 2 μL 3 2 μL 2 μL — 4 2 μL — — 5 — 2 μL 2μL

Relative Light Unit Reaction Tube A Tube B Avg. Light 1 18.2 22.1 20.152 1.4 1.4 1.4 3 4.2 3.8 4 4 2.1 1.8 1.95 5 13.1 15.8 14.45

In order to demonstrate the transfer of phosphate from dATP to ADP toform ATP, the reactions, as shown below, were assembled in duplicate intwenty microliters of PRPP Synthetase Buffer (above). They were thenincubated at 37° C. for 34 minutes. The added components had thefollowing formulations: 2.3×10⁻²M ADP in 10 mM Tris-Cl pH 7.3; 1000×dilution of NDPK (Sigma #N0379) at 10 U/μL (final concentration 0.01U/μL). The tubes were then incubated for an additional 60 minutes at 37°C., 10 ng luciferase added, and the light output measured using aTurner® TD-20e Luminometer. The data are presented in the table below.These data indicate that the dATP produced by the PRPP Synthetasereaction can be transferred to ADP by the action of NDPK to produce ATP.Reactions Assembled Reaction dAMP PRPP PRPP Syn ADP NDPK 1 2 μL 2 μL 2μL 2 μL 2 μL 2 2 μL 2 μL 2 μL — — 3 2 μL 2 μL 2 μL 2 μL — 4 2 μL 2 μL 2μL — 2 μL 5 — 2 μL 2 μL 2 μL 2 μL

Light Units Reaction Tube A Tube B 1 812.1 839.3 2 19.2 37.5 3 53.6 52.64 168.4 173.1 5 43.6 38.9

EXAMPLE 85 Digestion of PhiX 174 HinF1 Fragments

Polynucleotides encountered in nature are often double stranded. The DNAfragments generated by digestion of PhiX 174 DNA using endonucleaseHinFI are double-stranded DNA fragments of various sizes. In order totest whether double stranded DNA could be detected, PhiX 174 DNA wasdirectly used as a target nucleic acid substrate or digested withnucleases to produce nucleotides that could be converted to nucleosidetriphosphates as in previous Examples.

The following conditions were used to digest DNA fragments frombacteriophage PhiX 174. The following materials were placed in three 1.5mL polypropylene tubes: 50 μL of PhiX 174 HinFI fragments (PromegaG175A, Lot #773603); 40 μL 5 mM MgSO₄; 5 μL Exo III buffer (10×)(Promega E577B, 4853216), and 5 μL Nanopure water. Fifty microliters TEbuffer and 40 μL 5 mM MgSO₄; 5 μL ExoIII buffer (10×) and 5 μL Nanopurewater were added to one sample. Two of the samples containing PhiX 174DNA were further treated with 2 μL Exo III (Promega M181A, 5512708) andthe tubes placed in a 37° C. water bath for 60 minutes. ExoIII was alsoadded to the sample without DNA and the sample incubated at 37° C. 60minutes.

At this time, 800 μL Nanopure water and 100 μL (10×) S1 Nuclease Buffer(Promega, M577A, Lot #6748605) were added to all samples. Threemicroliters S1 nuclease (Promega, E576B, Lot #789881) were then added toall samples. All samples were incubated at 37° C. for 30 minutes.

Two hundred microliters from each of the three tubes containing DNA werediluted with 300 μL 1× TE Buffer and the absorbance read at 260 nm usinga Beckman DU 650 spectrophotometer. The readings recorded were: tube one(no nuclease addition), 0.3073; tube two (treatment with Exo III),0.5495; tube three (treatment with Exo III and S1 ), 0.5190. Theincreased absorbance values of the tubes treated with nuclease indicatethat the polymer was digested. These digests were subsequently used inother studies (see Example 86, below).

EXAMPLE 86 Detection of PhiX 174 HinF1 Fragments Using Nucleases, PRPPSynthetase, NDPK

This example demonstrates the detection of DNA by digestion of thepolymer to nucleoside monophosphates using nucleases, transformation ofthe nucleoside monophosphates to nucleoside triphosphates using PRPPSynthetase and PRPP along with transformation of ADP to ATP using thenucleoside triphosphates generated by the action of PRPP Synthetase, anddetection of the ATP using luciferase. A sample of deoxynucleotide (Poly(dA)) was prepared as described in Example 85. Different amounts ofdeoxynucleotide were used in the reactions as presented in Table 30.

The following additions were made to each reaction: 2 μL PRPP, 2 μL PRPPSynthetase, and 20 μL PRPP Synthetase buffer. The reactions proceeded at37° C. for 28 minutes, at which time the reactions were transferred to100 μl LAR Buffer containing 2 μL ADP and 2 μL NDPK. This secondreaction was permitted to proceed at room temperature for 20 minutes.The amount of ATP produced was measured by the addition of 10 ng ofluciferase followed by measuring light output with a luminometer. Thedata are presented in table below. These data show that this combinationof enzymes permitted detection of DNA. Reaction Nucleotide Amount InReaction Light Units 1 dAMP 200 ng, 600 pmoles 1018 2 dAMP  20 ng, 60pmoles 636 3 dAMP  2 ng, 6 pmoles 178 4 dAMP 200 pg, 600 fmoles 83 5none zero ng 69 6 PhiX 174 only 100 ng (=300 pmoles 46 dNMP; about 75pmoles dAMP) 7 PhiX 174 + ExoIII 100 ng 472 8 PhiX 174 + Exo + S1 100 ng448 9 No DNA + Exo + S1 zero ng 55

EXAMPLE 87 NDPK Transformation of ADP To ATP Using Deoxynucleotides

Luciferase can detect ATP at much lower concentrations than dATP orother nucleotides. By using dNTPs to generate ATP, an increase insensitivity results. In this experiment, the ability of enzymes totransfer the terminal phosphate of dNTPs to ADP, forming ATP and dNDPs,was analyzed.

Reactions were assembled which contained 100 μL LAR Buffer, 10 ngluciferase in the presence or absence of dNTPs (1 μM final concentrationwhen added), and 10 units NDPK (Sigma #N0379, Lot #127F81802). Thereactions were assembled with the exception of luciferase and incubatedfor 15 minutes at room temperature. Luciferase was added and lightoutput of the reactions was measured immediately using a Turner TD-20eLuminometer. The light output values measured are provided in the datatable below. These data confirm that NDPK is capable of transferring thephosphate from nucleoside triphosphates to ADP to form ATP, which can bedetected using luciferase. Data Table Tube # dNTP ADP NDPK ATP LightUnits 1 — + + 883 2 — − + + 15361 3 — + − 543 4 — − − + 21970 5 dATP + +13356 6 dATP − + 151 7 dCTP + + 13007 8 dCTP − + 6.9 9 dGTP + + 13190 10dGTP − + 7.3 11 TTP + + 19230 12 TTP − + 9.0

EXAMPLE 88 NDPK Transformation of ADP to ATP Using NDPK and ATP Analogs

Some enzymes that may be used to transform nucleotides show specificityfor adenosine nucleotides as phosphate donors. Adenosine nucleotides arenot used as high energy phosphate donors for these converting enzymes ifa luciferase detection system is to be utilized. This is because lightis generated by luciferase from the added adenosine nucleotide. However,the converting enzymes can be utilized if an analog of adenosine isidentified that can be used by the converting enzymes but not byluciferase. This example indicates how such analogs can be analyzed fortheir ability to be used by converting enzymes but not by luciferase.

Approximately 5 mg ATP (Sigma A9187, Lot #36H7808), α,βmethyleneadenosine 5′-Triphosphate (AMP-CPP) (Sigma M6517, Lot #96H7813)and β,γ methylene adenosine 5′-triphosphate (AMP-PCP) (Sigma M7510, Lot#34H7840) were diluted in Tris-Cl, 10 mM, pH 7.5. The absorbance of a1:100 dilution of these solutions into 50 mM Tris-Cl, pH 7.5 was read at259 nm using a Beckman DU650 Spectrophotometer. The absorbances wereused to determine the concentration of these solutions using a molarextinction coefficient of 15.4×10³ Molar. Recombinant luciferase wasdiluted into CCLR containing 1 mg/mL BSA to a concentration of 2.5ng/μL. When the reactions were assembled, 2 μL of luciferase were addedfrom the 2.5 ng/μL stock solution and the light emission of thesolutions were immediately read using a Turner TD-20e Luminometer. Thedata are provided in Table below. Reaction LAR ATP AMP-CPP* AMP-PCP* #rxns Avg. 1 50 μL — — — 3 426.4 2 50 μL 4 μM — — 7 5762 3 50 μL —  552μM — 2 349.2 4 50 μL 4 μM  552 μM — 2 5072.5 5 50 μL — 5.52 μM — 2 465.86 50 μL 4 μM 5.52 μM — 2 5843.5 7 50 μL — 55.2 nM — 2 429.8 8 50 μL 4 μM55.2 nM — 2 4152 9 50 μL — — 1.14 mM 2 260.35 10 50 μL 4 μM — 1.14 mM 23735.5 11 50 μL — — 11.4 μM 2 431.25 12 50 μL 4 μM — 11.4 μM 2 5930 1350 μL — —  114 nM 2 389.35 14 50 μL 4 μM  114 nM 2 6093.5*Final concentration in the reaction, solution produced by addition of 5μL of a more concentrated stock solution.

Micromolar solutions of these ATP analogs do not produce light abovethat of reactions containing no added nucleotide and do not greatlylower the light output of reactions containing low levels of ATP fromthe values seen in the absence of these analogs. These analogs do notinhibit luciferase and are not utilized by luciferase. Thus, these dataindicate that these analogs can be analyzed for their ability to be usedwith enzymes for the transformation of nucleotides.

The following reactions were performed to determine if either AMP-CPP orAMP-PCP could be used by NDPK. All reactions were assembled in duplicateand incubated at room temperature for 20 minutes. Ten nanograms ofluciferase were added and the light output of the reactions immediatelymeasured using a Turner TD-20e luminometer. The data are provided inTable below. These data demonstrate that the analog AMP-CPP is utilizedby the enzyme NDPK as a substrate to generate ATP from ADP. The valuesseen with AMP-CPP, ADP and NDPK present are substantially higher thanthose seen for ADP alone, ADP and NDPK without AMP-CPP and NDPK alone.Analogous experiments can be performed to test other enzymes for theirability to use nucleotide substrates in a similar fashion. AMP- AMP- CPPPCP LAR- ADP (2 × (2 × Reaction CoA (2 × 10⁻⁴M) NDPK 10⁻⁵M) 10⁻⁵M) Avg.1 100 μL — — — — 0.21 2 100 μL 0.5 μL — — — 60.23 3 100 μL 0.5 μL 1 μL —— 59.77 4 100 μL 0.5 μL 1 μL 5 μL — 617.95 5 100 μL — 1 μL 5 μL — 1.81 6100 μL 0.5 μL 1 μL — 5 μL 69.35 7 100 μL — 1 μL — 5 μL 0.03 8 100 μL — 1μL — — 0.05

EXAMPLE 89 Interrogation with a Self-Annealing Primer II

This example and FIG. 2 illustrate use of a different type ofoligonucleotide probe, a “REAPER™” probe in a process of this invention.This example demonstrates a method for eliminating the need for adding aprobe specific to the interrogation site to the interrogation reaction.

Here, the oligonucleotide first probe (SEQ ID NO:287), at its 3′-end,anneals to the target strand (SEQ ID NO:286) at a position downstream of(3′ to) the interrogation position in the target strand (FIG. 2A). Theprobe has at its 5′-end an unannealed region of nucleotides includingabout 5 to about 20 nucleotides that are identical to a region on thetarget strand including the interrogation position. This region ofidentity is present in the same orientation on both the target and theprobe strands.

The annealed 3′-end of the probe is then extended through theinterrogation position of the target strand forming what is referred toas a first extended probe and an extended first hybrid as is illustratedin FIG. 2B (SEQ ID NO:288). The extended first hybrid is denatured and asecond probe (SEQ ID NO:289) is annealed to the first extended probe toform a second hybrid. This second probe is complementary to the firstextended probe strand at a region downstream of the interrogationposition on the first extended probe strand (FIG. 2C).

The second probe is then extended and a second extended hybrid is formedas illustrated in FIG. 2D. The second extended hybrid is comprised ofthe first extended probe and second extended probe (SEQ ID NO:290).

The strands of the second extended hybrid are denatured and permitted torenature to form a hairpin structure. Upon hairpin formation, the firstextended probe forms a hairpin structure that has a 3′-overhang, whereasthe second extended probe forms a hairpin structure that contains a5′-overhang that provides a substrate for depolymerization. The secondextended probe strand is then depolymerized and the analytical outputobtained as described elsewhere herein. The analytical output determinesthe presence or absence of the original target strand or of a particularbase in the original target strand as is also discussed elsewhereherein.

SEQ ID NO:286 oligonucleotide is diluted to 1 mg/mL in water. Thissolution is labeled 286. SEQ ID NO:287 oligonucleotide is diluted to 1mg/mL in water and this solution is labeled 287. One microliter of eachsolution 286 and 287 is combined with 18 μL water. The solution isheated to 95° C. for 5 minutes then is cooled at room temperature for 10minutes to permit oligonucleotides of SEQ ID NOs:286 and 287 to anneal.

To this solution are added DNTP mixture to a final concentration of 0.25mM for each DNTP, 10× Klenow buffer to a final concentration of 1×, and5 U of Klenow enzyme. The tube with these components is incubated at 37°C. for 30 minutes. The extended first hybrid DNA so formed (containingSEQ ID NO: 288) is purified (Qiagen, Mermaid system) and eluted into 50μl of water.

To this solution of the purified extended first hybrid is added 1 μl SEQID NO: 289 oligonucleotide (1 mg/mL) as second probe. The solution isthen heated to 95° C. for 5 minutes and is cooled at room temperature topermit 289 and 288 to anneal as illustrated in FIG. 2C to form thesecond hybrid. To this solution are added a DNTP mixture to a finalconcentration of 0.25 mM for each dNTP, 10× Klenow buffer to a finalconcentration of 1×, and 5 U of Klenow enzyme. The tube with thesecomponents is incubated at 37° C. for 30 minutes to form a secondextended hybrid that contains a second extended probe (oligonucleotideSEQ ID NO: 290).

The SEQ ID NO: 290/288 second extended hybrid DNA (FIG. 2D) formed ispurified (Qiagen, Mermaid system) to separate the extended hybrid fromthe unreacted dNTPs and eluted into 50 μl water. (Alternatively, theoriginal 287 oligo is biotinylated at it's 5′-end and this biotin isthen also present in strand of SEQ ID NO: 288. This biotinylated strand288 is then denatured from strand 290 and removed from the solution withstreptavidin coated paramagnetic particles according to themanufacturer's instructions (Promega, Z5481) and the 290 hairpinstructure is allowed to form as below).

This hybrid solution is then heated to 95° C. for 5 minutes diluted to100 μl with water and is cooled on ice for 10 minutes to permit hairpinstructure formation.

The following master mix is assembled and mixed. Component Amount 10 ×DNA Pol Buffer   200 μL (Promega, M195A) Klenow exo- (1 U/μL)  12.5 μL(Promega M218B) 40 mM Sodium Pyrophosphate   25 μL (Promega C350B) NDPK(1 U/μL)   10 μL 10 uM ADP (Sigma A5285)   20 μL Water 732.5 μL

Twenty microliters of this master mix are added to 20 μL of the abovehairpin-containing solutions after cooling, and the resulting mixturesare heated at 37° C. for 15 minutes. After this incubation, duplicate 4μL samples of the solution are removed, added to 100 μL of L/L Reagent(Promega, F202A) and the light produced by the reaction is measuredimmediately using a Turner® TD20/20 luminometer. A positive analyticaloutput at levels over background (no enzyme) indicates that a matchedbase was present at the 3′-terminus of the hairpin structure and thisfurther indicates the presence of the target strand, and for thisparticular example, it also indicates the presence of a G base at theinterrogation position of the target. 5′CCCGGAGAGACCTCCTTAAGGGGCCATATTATT SEQ ID NO: 286TCGTCGATTCCAGTGTTGGCCAAACGGAT 3′ 5′ GGGGCCATATTATTTCGCCGTTTGGCCAACACTSEQ ID NO: 287 GGAATCGA 3′ 5′ GGGGCCATATTATTTCGCCGTTTGGCCAACACT SEQ IDNO: 288 GGAATCGACGAAATAATATGGCCCCTTAAGGAGGTC TCTCCGGG 3′ 5′CCCGGAGAGACCTCCT 3′ SEQ ID NO: 289 5′ CCCGGAGAGACCTCCTTAAGGGGCCATATTATTSEQ ID NO: 290 TCGTCGATTCCAGTGTTGGCCAAACGGCGAAATAAT ATGGCCCC 3′

From the foregoing, it will be observed that numerous modifications andvariations can be effected without departing from the true spirit andscope of the present invention. It is to be understood that nolimitation with respect to the specific examples presented is intendedor should be inferred. The disclosure is intended to cover by theappended claims modifications as fall within the scope of the claims.

1. A method for determining the presence or absence of a predeterminednucleic acid target sequence in a nucleic acid sample that comprises thesteps of: (A) providing a treated sample that may contain saidpredetermined nucleic acid target sequence hybridized with a nucleicacid probe that includes an identifier nucleotide in the 3′-terminalregion; (B) admixing the treated sample with a depolymerizing amount ofan enzyme whose activity is to release one or more nucleotides from the3′-terminus of a hybridized nucleic acid probe to form a treatedreaction mixture; (C) maintaining the treated reaction mixture for atime period sufficient to permit the enzyme to depolymerize hybridizednucleic acid and release identifier nucleotides therefrom; and (D)analyzing for the presence of released identifier nucleotides to obtainan analytical output, the analytical output indicating the presence orabsence of said nucleic acid target sequence.
 2. The method according toclaim 1 wherein said identifier nucleotide is a nucleoside triphosphate.3. The method according to claim 1 wherein said analytical output isobtained by luminescence spectroscopy.
 4. The method according to claim1 wherein said analytical output is obtained by fluorescencespectroscopy.
 5. The method according to claim 4 wherein said releasedidentifier nucleotide includes a fluorescent label.
 6. The methodaccording to claim 5 wherein said identifier nucleotide is fluorescentlylabeled after release from said hybrid.
 7. The method according to claim1 wherein said analytical output is obtained by mass spectrometry. 8.The method according to claim 7 wherein said released identifiernucleotide includes a fluorescent label.
 9. The method according toclaim 7 wherein said identifier nucleotide is fluorescently labeledafter release from said hybrid.
 10. The method according to claim 1wherein said analytical output is obtained by absorbance spectroscopy.11. The method according to claim 1 including the further steps offorming said treated sample by (a) admixing a sample to be assayed withone or more nucleic acid probes to form a hybridization composition,wherein the 3′-terminal region of said nucleic acid probes (i) hybridizewith partial or total complementarity to said nucleic acid targetsequence when that sequence is present in the sample and (ii) include anidentifier nucleotide; (b) maintaining said hybridization compositionfor a time period sufficient to form a treated sample that may containsaid one predetermined nucleic acid target sequence hybridized with anucleic acid probe.
 12. The method according to claim 1 wherein saidnucleic acid sample is obtained from a biological sample.
 13. The methodaccording to claim 12 wherein said predetermined nucleic acid targetsequence is a microbial or viral nucleic acid.
 14. The method accordingto claim 13 wherein said predetermined nucleic acid target sequence is aviral nucleic acid and the magnitude of the analytical output from apredetermined amount of said biological fluid provides a measure of theviral load in the biological sample.
 15. The method according to claim 1wherein said nucleic acid sample is obtained from a food source.
 16. Themethod according to claim 15 wherein said food source is a plant. 17.The method according to claim 16 wherein said predetermined nucleic acidtarget sequence is a sequence non-native to the genome of said plant.18. The method according to claim 17 wherein said sequence non-native tothe genome of said plant is a transcription control sequence.
 19. Themethod according to claim 18 wherein said transcription control sequenceis that of the 35S promoter or the NOS terminator.
 20. The methodaccording to claim 11 including the further steps of preparing a nucleicacid sample to be assayed by amplifying a nucleic acid of interest froma crude nucleic acid sample.
 21. A method for determining the presenceor absence of at least one predetermined nucleic acid target sequence ina nucleic acid sample that comprises the steps of: (A) admixing a sampleto be assayed with one or more nucleic acid probes to form ahybridization composition, wherein the 3′-terminal region of saidnucleic acid probes (i) hybridizes with partial or total complementarityto at least one said predetermined nucleic acid target sequence whenthat sequence is present in the sample and (ii) includes an identifiernucleotide; (B) maintaining said hybridization composition for a timeperiod sufficient to form a treated sample that may contain saidpredetermined nucleic acid target sequence hybridized with a nucleicacid probe; (C) admixing the treated sample with a depolymerizing amountof an enzyme whose activity is to release one or more nucleotides fromthe 3′-terminus of a hybridized nucleic acid probe to form a treatedreaction mixture; (D) maintaining the treated reaction mixture for atime period sufficient to permit the enzyme to depolymerize hybridizednucleic acid and release identifier nucleotides therefrom; and (E)analyzing for the presence of released identifier nucleotides to obtainan analytical output, the analytical output indicating the presence orabsence of at least one said nucleic acid target sequence.
 22. Themethod according to claim 21 wherein said identifier nucleotide is anucleoside triphosphate.
 23. The method according to claim 21 whereinsaid analytical output is obtained by luminescence spectroscopy.
 24. Themethod according to claim 21 wherein said analytical output is obtainedby fluorescence spectroscopy.
 25. The method according to claim 24wherein said released identifier nucleotide includes a fluorescentlabel.
 26. The method according to claim 25 wherein said identifiernucleotide is fluorescently labeled after release from said hybrid. 27.The method according to claim 21 wherein said analytical output isobtained by mass spectrometry.
 28. The method according to claim 27wherein said released identifier nucleotide includes a fluorescentlabel.
 29. The method according to claim 28 wherein said identifiernucleotide is fluorescently labeled after release from said hybrid. 30.The method according to claim 21 wherein said analytical output isobtained by absorbance spectroscopy.
 31. The method according to claim21 wherein said sample contains a plurality of predetermined nucleicacid target sequences and is admixed with a plurality of said nucleicacid probes.
 32. The method according to claim 31 wherein the analyticaloutput obtained when one of said nucleic acid probes hybridizes withpartial complementarity to one target nucleic acid sequence is greaterthan the analytical output when all of the nucleic acid probes hybridizewith total complementarity to their respective nucleic acid targetsequences.
 33. The method according to claim 31 wherein the analyticaloutput obtained when one of said nucleic acid probes hybridizes withpartial complementarity to one target nucleic acid sequence is less thanthe analytical output when all of the nucleic acid probes hybridize withtotal complementarity to their respective nucleic acid target sequences.34. The method according to claim 31 wherein the analytical outputobtained when one of said nucleic acid probes hybridizes with totalcomplementarity to one nucleic acid target sequence is greater than theanalytical output when all of the nucleic acid probes hybridize withpartial complementarity to their respective nucleic acid targetsequences.
 35. The method according to claim 31 wherein the analyticaloutput obtained when one of said nucleic acid probes hybridize withtotal complementarity to one target nucleic acid sequence is less thanthe analytical output when all of the nucleic acid probes hybridize withpartial complementarity to their respective nucleic acid targetsequences.
 36. The method according to claim 21 wherein said enzymewhose activity is to release nucleotides is a template-dependentpolymerase that, in the presence of pyrophosphate ions, depolymerizeshybridized nucleic acids whose bases in the 3′-terminal region arematched with total complementarity.
 37. The method according to claim 21wherein said enzyme whose activity is to release nucleotides exhibits a3→5′-exonuclease activity, depolymerizing hybridized nucleic acidshaving one or more mismatched bases in the 3′-terminal region of thehybridized probe.
 38. A method for determining the presence or absenceof a specific base in a nucleic acid target sequence in a sample to beassayed that comprises the steps of: (A) admixing a sample to be assayedwith one or more nucleic acid probes to form a hybridizationcomposition, wherein the 3′-terminal region of at least one of saidnucleic acid probes (i) is substantially complementary to said nucleicacid target sequence and comprises at least one predetermined nucleotideat an interrogation position, and (ii) includes an identifiernucleotide, and wherein said nucleic acid target sequence comprises atleast one specific base whose presence or absence is to be determined(B) maintaining said hybridization composition for a time periodsufficient to form a treated sample, wherein said interrogation positionof the probe is a nucleotide that is aligned with said specific base tobe identified in said target sequence, when present, so that basepairing can occur; (C) admixing the treated sample with an enzyme whoseactivity is to release one or more nucleotides from the 3′-terminus of ahybridized nucleic acid probe to depolymerize the hybrid and form atreated reaction mixture; (D) maintaining the treated reaction mixturefor a time period sufficient to release an identifier nucleotidetherefrom; and (E) analyzing for the presence or absence of releasedidentifier nucleotide to obtain an analytical output that indicates thepresence or absence of said specific base to be identified.
 39. Themethod according to claim 38 wherein the identifier nucleotide is at theinterrogation position.
 40. The method according to claim 38 whereinsaid analytical output is obtained by fluorescence spectroscopy.
 41. Themethod according to claim 40 wherein said identifier nucleotide isfluorescently labeled after release from said hybrid.
 42. The methodaccording to claim 38 wherein said analytical output is obtained by massspectrometry.
 43. The method according to claim 40 wherein said releasedidentifier nucleotide includes a fluorescent label.
 44. The methodaccording to claim 42 wherein said released identifier nucleotideincludes a fluorescent label.
 45. The method according to claim 38,wherein said nucleic acid target sequence is selected from the groupconsisting of deoxyribonucleic acid and ribonucleic acid.
 46. The methodaccording to claim 45, further comprising a first probe, a second probe,a third probe and a fourth probe.
 47. The method according to claim 46,wherein said interrogation position of said first probe comprises anucleic acid residue that is a deoxyadenosine or adenosine residue, saidinterrogation position of said second probe comprises a nucleic acidresidue that is a deoxythymidine or uridine residue, said interrogationposition of said third probe comprises a nucleic acid residue that is adeoxyguanosine or guanosine residue, and said fourth nucleic acid probecomprises a nucleic acid residue that is a deoxycytosine or cytosineresidue.
 48. The method according to claim 38 wherein said sample to beassayed comprises a plurality of nucleic acid target sequences in whichthe presence or absence of a plurality of specific bases isinterrogated.
 49. The method according to claim 48 wherein theanalytical output obtained when one of said nucleic acid probeshybridizes with partial complementarity to one target nucleic acidsequence is greater than the analytical output when all of the nucleicacid probes hybridize with total complementarity to their respectivenucleic acid target sequences.
 50. The method according to claim 48wherein the analytical output obtained when one of said nucleic acidprobes hybridizes with partial complementarity to one target nucleicacid sequence is less than the analytical output when all of the nucleicacid probes hybridize with total complementarity to their respectivenucleic acid target sequences.
 51. The method according to claim 48wherein the analytical output obtained when one of said nucleic acidprobes hybridizes with total complementarity to one nucleic acid targetsequence is greater than the analytical output when all of the nucleicacid probes hybridize with partial complementarity to their respectivenucleic acid target sequences.
 52. The method according to claim 48wherein the analytical output obtained when one of said nucleic acidprobes hybridize with total complementarity to one target nucleic acidsequence is less than the analytical output when all of the nucleic acidprobes hybridize with partial complementarity to their respectivenucleic acid target sequences.
 53. The method according to claim 38wherein said enzyme whose activity is to release nucleotides is atemplate-dependent polymerase that, in the presence of pyrophosphateions, depolymerizes hybridized nucleic acids whose bases in the3′terminal region of the probe are matched with total complementarity.54. The method according to claim 38 wherein said enzyme whose activityis to release nucleotides exhibits a 3′→5′-exonuclease activity,depolymerizing hybridized nucleic acids having one or more mismatchedbases at the 3′-terminus of the hybridized probe.
 55. A method fordetermining the presence or absence of a first nucleic acid target in anucleic acid sample containing that target or a substantially identicalsecond target that comprises the steps of: (A) admixing said sample tobe assayed with one or more nucleic acid probes to form a hybridizationcomposition, wherein said first and second nucleic acid targets comprisea region of sequence identity except for at least a single nucleotide ata predetermined position that differs between the targets, and whereinsaid nucleic acid probe (i) is substantially complementary to saidnucleic acid target region of sequence identity and comprises at leastone nucleotide at an interrogation position, said interrogation positionof the probe being aligned with said predetermined position of a targetwhen a target and probe are hybridized and (ii) includes an identifiernucleotide in the 3′-terminal region; (B) maintaining said hybridizationcomposition for a time period sufficient to form a treated samplewherein the nucleotide at said interrogation position of said probe isaligned with the nucleotide at said predetermined position of saidtarget in said region of identity; (C) admixing the treated sample witha depolymerizing amount an enzyme whose activity is to release one ormore nucleotides from the 3′-terminus of a hybridized nucleic acid probeto form a treated reaction mixture; (D) maintaining the treated reactionmixture for a time period sufficient to release identifier nucleotideand depolymerize said hybridized nucleic acid probe; and (E) analyzingfor the presence of released identifier nucleotide to obtain ananalytical output, said analytical output indicating the presence orabsence of said nucleotide at said predetermined region and thereby thepresence or absence of a first or second nucleic acid target.
 56. Themethod according to claim 55 wherein said analytical output is obtainedby fluorescence spectroscopy.
 57. The method according to claim 56wherein said identifier nucleotide is fluorescently labeled afterrelease from said hybrid.
 58. The method according to claim 56 whereinsaid analytical output is obtained by mass spectrometry.
 59. The methodaccording to claim 56 or 59 wherein said released identifier nucleotideincludes a fluorescent label.
 60. The method according to claim 56wherein said identifier nucleotide is a nucleoside triphosphate.
 61. Themethod according to claim 56 wherein said analytical output is obtainedby luminescence spectroscopy.
 62. The method according to claim 61wherein said analytical output is obtained by absorbance spectroscopy.63. The method according to claim 56 wherein said nucleic acid targetsequence is selected from the group consisting of deoxyribonucleic acidand ribonucleic acid.
 64. The method according to claim 56 furthercomprising a first probe and a second probe.
 65. The method according toclaim 65 wherein said sample to be assayed comprises a plurality firstnucleic acid targets and second substantially identical nucleic acidtargets.
 66. The method according to claim 66 wherein said first probecomprises a nucleotide at said interrogation position that iscomplementary to a first target nucleic acid at said predeterminedposition, and said second probe comprises a nucleotide at theinterrogation position that is complementary to a second target nucleicacid at said predetermined position.
 67. The method according to claim66 wherein the analytical output obtained when one of said nucleic acidprobes hybridizes with partial complementarity to one target nucleicacid sequence is greater than the analytical output when all of thenucleic acid probes hybridize with total complementarity to theirrespective nucleic acid target sequences.
 68. The method according toclaim 66 wherein the analytical output obtained when one of said nucleicacid probes hybridizes with partial complementarity to one targetnucleic acid sequence is less than the analytical output when all of thenucleic acid probes hybridize with total complementarity to theirrespective nucleic acid target sequences.
 69. The method according toclaim 66 wherein the analytical output obtained when one of said nucleicacid probes hybridizes with total complementarity to one nucleic acidtarget sequence is greater than the analytical output when all of thenucleic acid probes hybridize with partial complementarity to theirrespective nucleic acid target sequences.
 70. The method according toclaim 66 wherein the analytical output obtained when one of said nucleicacid probes hybridizes with total complementarity to one target nucleicacid sequence is less than the analytical output when all of the nucleicacid probes hybridize with partial complementarity to their respectivenucleic acid target sequences.
 71. The method according to claim 56wherein said enzyme whose activity is to release nucleotides is atemplate-dependent polymerase that, in the presence of pyrophosphateions, depolymerizes hybridized nucleic acids whose bases in the3′-terminal region are matched with total complementarity.
 72. Themethod according to claim 56 wherein said enzyme whose activity is torelease nucleotides exhibits a 3′→5′-exonuclease activity,depolymerizing hybridized nucleic acids having one or more mismatchedbases in the 3′-terminal region of the hybridized probe.
 73. A methodfor selectively detecting a poly(A)⁺ RNA that comprises the steps of:(A) admixing a sample to be assayed with an oligo(dT) probe to form ahybridization composition, wherein said oligo(dT) probe includes anidentifier nucleotide in the 3′-terminal region; (B) maintaining saidhybridization composition for a time period sufficient to form a treatedsample wherein said poly(A)⁺ RNA hybridizes to said oligo(dT) probe; (C)admixing the treated sample with an enzyme whose activity is to releaseof one or more nucleotides from the 3′-terminus of a nucleic acidhybrid, including the identifier nucleotide, to form a treated reactionmixture; (D) maintaining the treated reaction mixture for a time periodsufficient to release identifier nucleotides therefrom and depolymerizehybridized nucleic acid probe; and (E) analyzing for the presence ofreleased identifier nucleotide to obtain an analytical output, saidanalytical output indicating the presence of said poly(A)⁺ RNA.
 74. Themethod according to claim 74 wherein said identifier nucleotide is anucleoside triphosphate.
 75. The method according to claim 74 whereinsaid analytical output is obtained by luminescence spectroscopy.
 76. Themethod according to claim 74 wherein said analytical output is obtainedby fluorescence spectroscopy.
 77. The method according to claim 74wherein said identifier nucleotide includes a fluorescent label.
 78. Themethod according to claim 74 wherein said analytical output is obtainedby mass spectrometry.
 79. The method according to claim 78 wherein saidanalytical output is obtained by mass spectrometry.
 80. The methodaccording to claim 78 wherein said analytical output is obtained byabsorbance spectroscopy.
 81. The method according to claim 74 whereinsaid enzyme whose activity is to release nucleotides is atemplate-dependent polymerase that, in the presence of pyrophosphateions, depolymerizes hybridized nucleic acids whose 3′-terminal bases arecompletely matched.
 82. The method according to claim 74 wherein saidoligo(dT) probe is completely hybridized with said poly(A)⁺ mRNA.
 83. Amethod for determining the number of known sequence repeats present in anucleic acid target sequence in a nucleic acid sample that comprises thesteps of: (A) providing a plurality of separate treated samples, eachtreated sample containing a nucleic acid target sequence hybridized witha nucleic acid probe wherein (a) the nucleic acid target sequencecontains (i) a plurality of known sequence repeats and (ii) anon-repeated region downstream of the repeats, and (b) the nucleic acidprobe is one of a plurality of different probes wherein said probesdiffer in the number of complementary sequence repeats containedtherein, each nucleic acid probe containing (i) a plurality of sequencerepeats complementary to the known sequence repeat of alleles of thetarget nucleic acid, (ii) an identifier nucleotide in the 3′-terminalregion of the probe and (iii) a 5′-terminal locker sequence that iscomplementary to the non-repeated region of the target and comprises 1to about 20 nucleotides. (B) admixing each treated sample with adepolymerizing amount of an enzyme whose activity is to release one ormore nucleotides from the 3′-terminus of a hybridized nucleic acid probeto form a treated depolymerization reaction mixture; (C) maintaining thetreated depolymerization reaction mixtures for a time period sufficientto permit the enzyme to depolymerize hybridized nucleic acid probe andrelease identifier nucleotide therefrom; and (D) analyzing the samplesfor the presence of released identifier nucleotide to obtain ananalytical output indicative of the number of sequence repeats presentin said nucleic acid target sequence.
 84. The method according to claim84 wherein said nucleic acid sample comprises two nucleic acid targetsequences representing alleles and is homozygous with respect to thenumber of sequence repeats in the two alleles.
 85. The method accordingto claim 84 wherein said nucleic acid sample comprises two nucleic acidtarget sequences representing alleles and is heterozygous with respectto the number of sequence repeats in the two alleles.
 86. The methodaccording to claim 84 wherein said identifier nucleotide is a nucleotidethat is part of the repeated sequence.
 87. The method according to claim76 wherein said identifier nucleotide of the probe sequence iscomplementary to a non-repeating sequence located 3′ to the repeatedsequences of the target nucleic acid.
 88. The method according to claim88 wherein said identifier nucleotide is present in a sequencecontaining 1 to about 20 nucleic acids that is complementary to anon-repeating sequence located 3′ to the repeated sequences of thetarget nucleic acid.
 89. The method according to claim 84 wherein arepeated known sequence present in a nucleic acid target sequence has alength of 2 to about 24 bases per repeat.
 90. A one-pot method fordetermining the presence or absence of at least one predeterminednucleic acid target sequence in a nucleic acid sample that comprises thesteps of: (A) admixing a treated sample that may contain saidpredetermined nucleic acid target sequence hybridized to a nucleic acidprobe whose 3′-terminal region is completely complementary to saidpredetermined nucleic acid target sequence and includes an identifiernucleotide with (i) a depolymerizing amount of an enzyme whose activityin the presence of pyrophosphate is to release identifier nucleotide asa nucleoside triphosphate from the hybridized nucleic acid probe, (ii)adenosine 5′ diphosphate, (iii) pyrophosphate and (iv) NDPK to form atreated reaction mixture; (b) maintaining the treated reaction mixtureat a temperature of about 25 to about 80 degrees C. for a time periodsufficient to permit the enzyme to depolymerize hybridized nucleic acidprobe, release an identifier nucleotide in the 3′-terminal region as anucleoside triphosphate and to convert said nucleoside triphosphate andsaid adenosine 5′ diphosphate to adenosine 5′ triphosphate; and (d)analyzing for the presence of adenosine 5′ triphosphate to obtain ananalytical output, the analytical output indicating the presence orabsence of at least one said nucleic acid target sequence.
 91. Themethod according to claim 91 wherein said analytical output is obtainedby luminescence spectroscopy.
 92. The method according to claim 91including the further steps of forming said treated sample by (a)admixing a sample to be assayed with one or more nucleic acid probes toform a hybridization composition, wherein the 3′-terminal region of saidnucleic acid probe (i) hybridizes with partial or total complementarityto a nucleic acid target sequence when that sequence is present in thesample and (ii) includes an identifier nucleotide; (b) maintaining saidhybridization composition for a time period sufficient to form a treatedsample that may contain said one predetermined nucleic acid targetsequence hybridized with a nucleic acid probe.
 93. The method accordingto claim 91 wherein said depolymerizing enzyme maintains activity at60-90° C.
 94. The method according to claim 91 wherein saiddepolymerizing enzyme is selected from the group consisting of the Tnetriple mutant DNA polymerase, Bst DNA polymerase, Ath DNA polymerase,Taq DNA polymerase and Tvu DNA polymerase.
 95. The method according toclaim 91 wherein said NDPK is that encoded by Pyrococcus furiosis.
 96. Amethod for enhancing the discrimination of analytical output in thedetermination of the presence or absence of a predetermined targetnucleic acid sequence in a nucleic acid sample that comprises the stepsof: (A) providing a plurality of separate treated samples, each samplecontaining (a) a nucleic acid that may contain said predeterminednucleic acid target sequence, said nucleic acid target sequence beinghybridized when present with (b) a nucleic acid probe, a first probe ofa first treated sample comprising (i) a 3′-terminal region sequence thatis complementary to said nucleic acid target sequence and includes anidentifier nucleotide that is complementary to a first predeterminednucleotide of said nucleic acid target sequence and (ii) a secondsequence otherwise complementary to said nucleic acid target sequenceexcept for a second predetermined nucleotide located 2 to about 10nucleotides upstream from the 3′-terminus of said probe that is notcomplementary to a second nucleotide of said nucleic acid targetsequence, and a second probe of a second treated sample comprising (i) a3′-terminal sequence that is complementary to said nucleic acid targetsequence except for an identifier nucleotide that is not complementaryto said first-named predetermined nucleotide of said nucleic acid targetsequence and (ii) a second sequence otherwise complementary to saidnucleic acid target sequence except for said second predeterminednucleotide located 3 to about 10 nucleotides upstream from the3′-terminus of said probe that is not complementary to a secondpredetermined nucleotide of said nucleic acid target sequence; (B)admixing each treated sample with a depolymerizing amount of an enzymewhose activity is to release one or more nucleotides from the3′-terminus of a hybridized nucleic acid probe to form a treatedreaction mixture; (C) maintaining the treated reaction mixtures for atime period sufficient to permit the enzyme to depolymerize hybridizednucleic acid probe and release an identifier nucleotide; and (D)analyzing the samples for the presence of released identifier nucleotideto obtain an analytical output, the ratio of the analytical output fromthe sample containing the first probe relative to that of the secondprobe being enhanced compared to the ratio of the analytical output froma similar sample containing a third probe of the same length having thesame identifier nucleotide and total complementarity to said nucleicacid target sequence relative to that of a fourth probe of the samelength whose identifier nucleotide is non-complementary to said firstpredetermined nucleotide and is otherwise totally complementary to saidtarget nucleic acid sequence.
 97. A method for determining whether thenucleic acid target sequence in a nucleic acid sample is an allele froma homozygous or heterozygous locus that comprises the steps of: (A)providing a plurality of separate treated samples, each samplecontaining (a) a nucleic acid target sequence hybridized with (b) anucleic acid probe, said nucleic acid target sequence being that of afirst allele, a second allele or a mixture of said first and secondalleles from a locus of interest of said nucleic acid target, saidalleles differing in sequence at an interrogation position, said nucleicacid probe containing an identifier nucleotide in the 3′-terminal regionthat is aligned at an interrogation nucleotide position of the targetsequence when said probe and target are hybridized; (B) admixing eachtreated sample with a depolymerizing amount of an enzyme whose activityis to release one or more nucleotides from the 3′-terminus of ahybridized nucleic acid probe to form a treated reaction mixture; (C)maintaining the treated reaction mixtures for a time period sufficientto permit the enzyme to depolymerize hybridized nucleic acid probe andan release identifier nucleotide; and (D) analyzing the samples for thepresence of released identifier nucleotide to obtain an analyticaloutput, the analytical output indicating whether the nucleic acid targetsequence in a nucleic acid sample is an allele from a homozygous or aheterozygous locus.
 98. The method of claim 98 wherein said analyzingcomprises analyzing the samples for the quantity of released identifiernucleotide to obtain an analytical output, the analytical outputindicating whether the nucleic acid target sequence in the nucleic acidsample is homozygous or heterozygous when compared to the analyticaloutput of an appropriate control.
 99. The method according to claim 98wherein said analytical output indicates which allele is present whenthe nucleic acid target sequence in a nucleic acid sample is homozygousat the locus of interest.
 100. The method according to claim 98 whereinsaid enzyme whose activity is to release nucleotides is atemplate-dependent polymerase that, in the presence of pyrophosphateions, depolymerizes hybridized nucleic acids whose bases in the3′-terminal region are completely complementary to bases of said nucleicacid target.
 101. The method according to claim 100 wherein saidanalytical output is obtained by luminescence spectroscopy.
 102. Amethod for determining the presence or absence of a nucleic acid targetsequence containing an interrogation position in a nucleic acid samplethat comprises the steps of: (A) providing a treated sample thatcontains a nucleic acid sample that may include said nucleic acid targetsequence hybridized with a nucleic acid probe that is comprised of threesections, (i) a first section that contains the probe 3′-terminal about10 to about 30 nucleotides that are complementary to the nucleic acidtarget sequence at positions beginning about 1 to about 30 nucleic acidsdownstream of said interrogation position of the target sequence, (ii) a5′-terminal region of about 10 to about 200 nucleic acids in length andhaving the identical sequence of said nucleic acid target sequence, and(iii) an optional third section that contains zero to about 50 nucleicacids that are not complementary to said nucleic acid sample, and; (B)extending said nucleic acid probe in a 3′ direction to form a secondprobe hybridized to the nucleic acid sample as a second hybrid; (D)denaturing said second hybrid to separate said second probe from saidnucleic acid target sequence; (E) renaturing said aqueous composition toform hairpin structures from said second probe; (F) admixing the hairpinstructure-containing composition with a depolymerizing amount of anenzyme whose activity is to release one or more nucleotides from the3′-terminus of a nucleic acid hybrid to form a treated reaction mixture;(G) maintaining the treated reaction mixture for a time periodsufficient to permit the enzyme to depolymerize hybridized nucleic acidand release one or more nucleotides from the 3′-terminus therefrom; and(H) analyzing for the presence of released identifier nucleotide toobtain an analytical output, the analytical output indicating thepresence or absence of said nucleic acid target sequence.
 103. A methodfor determining the presence or absence of a RNA target sequence in anucleic acid sample that comprises the steps of: (A) providing a treatedsample that may contain said predetermined RNA target sequencehybridized with a nucleic acid probe that includes an identifiernucleotide in the 3′-terminal region; (B) admixing the treated samplewith a depolymerizing amount of an enzyme whose activity is to releaseone or more nucleotides from the 3′-terminus of a hybridized nucleicacid probe to form a treated reaction mixture; (C) maintaining thetreated reaction mixture for a time period sufficient to permit theenzyme to depolymerize hybridized nucleic acid probe and releaseidentifier nucleotide therefrom; (D) forming a reaction mixture byadmixture of a second nucleic acid probe that hybridizes with saidfirst-named probe, said second probe containing a 3′-terminal regionidentifier nucleotide that can be released by the enzyme of step (B) butis not released unless one or more nucleotides have been released fromthe 3′-terminus of said first-named probe; (E) maintaining the reactionmixture for a time period sufficient to permit the enzyme todepolymerize hybridized nucleic acid from said second nucleic acid probeand release identifier nucleotide; and (F) analyzing for the presence ofreleased identifier nucleotide to obtain an analytical output, theanalytical output indicating the presence or absence of said RNA targetsequence.
 104. The method according to claim 104 wherein said secondnucleic acid probe is admixed with said treated sample and steps (D) and(E) are carried out together with steps (A) and (B).
 105. A method fordetermining the presence or absence of a restriction endonucleaserecognition sequence in a nucleic acid sample that comprises the stepsof: (A) providing a treated sample that may contain a hybridized nucleicacid target that is a cleaved restriction endonuclease recognitionsequence that includes an identifier nucleotide in the restrictionendonuclease recognition sequence; (B) admixing the treated sample witha depolymerizing amount of an enzyme whose activity is to release one ormore nucleotides from the 3′-terminus of a restriction endonucleaserecognition sequence to form a treated reaction mixture; (C) maintainingthe treated reaction mixture for a time period sufficient to permit theenzyme to depolymerize hybridized nucleic acid and release identifiernucleotide therefrom; and (D) analyzing for the presence of releasedidentifier nucleotide to obtain an analytical output, the analyticaloutput indicating the presence or absence of said restrictionendonuclease recognition sequence.
 106. The process of claim 106including the further steps of forming a treated sample by: (A)providing an endonuclease cleavage reaction solution comprising anucleic acid sample and a restriction endonuclease enzyme specific forthe restriction endonuclease recognition sequence; and (B) maintainingthe endonuclease cleavage reaction solution for a time period sufficientfor the restriction endonuclease enzyme to cleave the restrictionendonuclease recognition sequence to form a treated sample.
 107. Theprocess of claim 107 including the further step of amplifying a nucleicacid target sequence in a nucleic acid sample prior to providing saidrestriction endonuclease recognition sequence.
 108. The process of claim108 wherein said nucleic acid target sequence is amplified by thefurther steps of: (A) admixing a crude nucleic acid sample with PCRamplification primers that are complementary to regions upstream anddownstream of the nucleic acid target sequence and a template-dependentpolymerase to form an amplification sample mixture wherein either thenucleic acid target sequence or the PCR amplification primers includes arestriction endonuclease recognition sequence; (B) maintaining theamplification sample mixture for a time period sufficient to denaturethe nucleic acid target sequence to form a denatured amplificationreaction mixture; (C) annealing the denatured amplification reactionmixture for a time period sufficient for PCR amplification primers toanneal to the nucleic acid target sequence to form an amplificationreaction mixture; and (D) maintaining the amplification reaction mixturefor a time period sufficient to permit the template-dependent polymeraseto extend the nucleic acid from the PCR primers to form an amplifiednucleic acid sample.
 109. A method for determining the loss ofheterozygosity of a locus of an allele that comprises the steps of: (A)providing a plurality of separate treated samples, each samplecontaining (a) a nucleic acid target sequence hybridized with (b) anucleic acid probe, said nucleic acid target sequence being that of afirst allele or a mixture of said first allele and a second allele ofsaid nucleic acid target, said alleles differing in sequence at aninterrogation position, said nucleic acid probe containing a 3′-terminalregion that hybridizes to a region of said nucleic acid target sequencecontaining said interrogation nucleotide position when said probe andtarget are hybridized and an identifier nucleotide; (B) admixing eachtreated sample with a depolymerizing amount of an enzyme whose activityis to release one or more nucleotides from the 3′-terminus of ahybridized nucleic acid probe to form a treated reaction mixture; (C)maintaining the treated reaction mixtures for a time period sufficientto depolymerize hybridized nucleic acid probe and an release identifiernucleotide; and (D) analyzing the samples for the quantity of releasedidentifier nucleotide to obtain an analytical output, the analyticaloutput indicating whether the nucleic acid target sequence in a nucleicacid sample has lost heterozygosity at the locus of the allele.
 110. Themethod of claim 109 wherein the quantity of said released identifiernucleotide for said first allele is substantially less that the quantityof said released identifier nucleotide for said first allele of a knownheterozygous control, and the quantity of said released identifiernucleotide for said second allele is substantially similar to thequantity of said released identifier nucleotide for said second alleleof a known heterozygous control, indicating a loss of heterozygosity atthe locus of said first allele.
 111. The method of claim 109 wherein thequantity of said released identifier nucleotide for said second alleleis substantially less that the quantity of said released identifiernucleotide for said second allele of a known heterozygous control, andthe quantity of said released identifier nucleotide for said firstallele is substantially similar to the quantity of said releasedidentifier nucleotide for said first allele of a known heterozygouscontrol, indicating a loss of heterozygosity at the locus of said secondallele.
 112. The method according to claim 110 wherein said analyticaloutput is obtained by luminescence spectroscopy.
 113. The methodaccording to claim 110 wherein said analytical output is obtained byabsorbance spectrometry.
 114. The method according to claim 110 whereinsaid analytical output is obtained by fluorescence spectroscopy. 115.The method according to claim 113 wherein said released identifiernucleotide includes a fluorescent label.
 116. The method according toclaim 114 wherein said identifier nucleotide is fluorescently labeledafter release from said hybrid.
 117. The method according to claim 110wherein said analytical output is obtained by mass spectrometry. 118.The method according to claim 116 wherein said released identifiernucleotide includes a fluorescent label.
 119. The method according toclaim 117 wherein said identifier nucleotide is fluorescently labeledafter release from said hybrid.
 120. The method according to claim 110wherein said enzyme whose activity is to release nucleotides is atemplate-dependent polymerase that, in the presence of pyrophosphateions, depolymerizes hybridized nucleic acids whose bases in the3′-terminal region are completely complementary to bases of said nucleicacid target.
 121. The method according to claim 119 wherein the quantityof said released identifier nucleotide for said first allele issubstantially less than the quantity of said released identifiernucleotide for said first and second alleles, indicating a loss ofheterozygosity at the locus of said first allele.
 122. The methodaccording to claim 119 wherein the quantity of said released identifiernucleotide for said second allele is substantially less than thequantity of said released identifier nucleotide for said first andsecond alleles, indicating a loss of heterozygosity at the locus of saidsecond allele.
 123. A method for determining the presence of trisomy ofan allele that comprises the steps of: (A) providing a plurality ofseparate treated samples, each sample containing (a) a nucleic acidtarget sequence hybridized with (b) a nucleic acid probe, said nucleicacid target sequence being that of a first allele, a second allele or amixture of said first and second alleles of said nucleic acid target,said alleles differing in sequence at an interrogation position, saidnucleic acid probe containing a 3′-terminal region that hybridizes to aregion of said nucleic acid target sequence containing saidinterrogation nucleotide position when said probe and target arehybridized and an identifier nucleotide; (B) admixing each treatedsample with a depolymerizing amount of an enzyme whose activity is torelease one or more nucleotides from the 3′-terminus of a hybridizednucleic acid probe to form a treated reaction mixture; (C) maintainingthe treated reaction mixtures for a time period sufficient todepolymerize hybridized nucleic acid probe and an release identifiernucleotide; and (D) analyzing the samples for released identifiernucleotide to obtain an analytical output, the quantity of saidanalytical output relative to an analytical output of a control sampleindicating whether a trisomy is present in the nucleic acid targetsequence.
 124. The method of claim 122 wherein the ratio of the quantityof said released identifier nucleotide for said first and second alleleis about 3 to 0, compared to the ratio of the quantity of said releasedidentifier nucleotide for said first and second allele of a knownheterozygous control of about 1 to 1, indicating a trisomy at the locusof said first allele.
 125. The method of claim 122 wherein the ratio ofthe quantity of said released identifier nucleotide for said first andsecond allele is about 0 to 3, compared to the ratio of the quantity ofsaid released identifier nucleotide for said first and second allele ofa known heterozygous control of about 1 to 1, indicating a trisomy atthe locus of said second allele.
 126. The method of claim 122 whereinthe ratio of the quantity of said released identifier nucleotide forsaid first and second allele is about 2 to 1, compared to the ratio ofthe quantity of said released identifier nucleotide for said first andsecond allele of a known heterozygous control of about 1 to 1,indicating a trisomy having two copies of the locus of said first alleleand one copy of the locus of said second allele.
 127. The method ofclaim 122 wherein the ratio of the quantity of said released identifiernucleotide for said first and second allele is about 1 to 2, compared tothe ratio of the quantity of said released identifier nucleotide forsaid first and second allele of a known heterozygous control of about 1to 1, indicating a trisomy having one copy of the locus of said firstallele and two copies of the locus of said second allele.
 128. Themethod according to claim 122 wherein said analytical output is obtainedby luminescence spectroscopy.
 129. The method according to claim 122wherein said analytical output is obtained by absorbance spectrometry.130. The method according to claim 122 wherein said analytical output isobtained by fluorescence spectroscopy.
 131. The method according toclaim 125 wherein said released identifier nucleotide includes afluorescent label.
 132. The method according to claim 126 wherein saididentifier nucleotide is fluorescently labeled after release from saidhybrid.
 133. The method according to claim 122 wherein said analyticaloutput is obtained by mass spectrometry.
 134. The method according toclaim 128 wherein said released identifier nucleotide includes afluorescent label.
 135. The method according to claim 129 wherein saididentifier nucleotide is fluorescently labeled after release from saidhybrid.
 136. The method according to claim 122 wherein said enzyme whoseactivity is to release nucleotides is a template-dependent polymerasethat, in the presence of pyrophosphate ions, depolymerizes hybridizednucleic acids whose bases in the 3′-terminal region are completelycomplementary to bases of said nucleic acid target.
 137. The methodaccording to claim 131 wherein the quantity of said released identifiernucleotide for said first allele is substantially greater than thequantity of said released identifier nucleotide of a homozygous control,indicating that said nucleic acid target sequence has a trisomy. 138.The method according to claim 131 wherein the quantity of said releasedidentifier nucleotide for said second allele is substantially greaterthan the quantity of said released identifier nucleotide of a homozygouscontrol, indicating that said nucleic acid target sequence has atrisomy.
 139. An isolated and purified nucleotide diphosphate kinase(NDPK) enzyme that exhibits higher NDPK activity at a temperature ofabout 50 to about 90 degrees C. relative to the NDPK activity at 37degrees C.
 140. The isolated and purified NDPK enzyme according to claim134 that comprises the amino acid sequence of SEQ ID NO:90.
 141. Theisolated and purified NDPK enzyme according to claim 134 whose DNAsequence is that of SEQ ID NO:91.
 142. A method for determining thepresence or absence of a nucleic acid target sequence, or a specificbase within the target sequence, in a nucleic acid sample, thatcomprises the steps of: (A) providing a treated sample that contains anucleic acid sample that may include a nucleic acid target sequencehybridized with a first nucleic acid probe as a first hybrid, said firstprobe being comprised of at least two sections, a first sectioncontaining the probe 3′-terminal about 10 to about 30 nucleotides thatare complementary to the target nucleic acid sequence at a positionbeginning about 5 to about 30 nucleotides downstream of the targetinterrogation position, a second section of the first probe containingabout 5 to about 30 nucleotides that are a repeat of the target sequencefrom the interrogation position to about 10 to about 30 nucleotidesdownstream of the interrogation position that does not hybridize to saidfirst section of the probe, and an optional third section of the probelocated between the first and second sections of the probe that is zeroto about 50 nucleotides in length and comprises a sequence that does nothybridize to either the first or second section of the probe; (B)extending the first hybrid in the treated sample at the 3′-end of thefirst probe, thereby extending the first probe past the interrogationposition and forming an extended first hybrid that includes aninterrogation position; (C) denaturing an aqueous composition of theextended first hybrid to separate the two nucleic acid strands and forman aqueous composition containing a separated target nucleic acid and aseparated extended first probe; (D) annealing to the extended firstprobe a second probe that is about 10 to about 30 nucleotides in lengthand is complementary to the extended first probe at a position beginningabout 5 to about 2000 nucleotides downstream of the interrogationposition in the extended first probe, thereby forming a second hybrid;(E) extending the second hybrid at the 3′-end of the second probe untilthat extension reaches the 5′-end of the extended first probe, therebyforming a second extended hybrid containing a second extended probewhose 3′-region includes an identifier nucleotide; (F) denaturing anaqueous composition of the extended second hybrid to separate the twonucleic acid strands and form an aqueous composition containingseparated extended first and second probes; (G) cooling the aqueouscomposition to form a hairpin structure from the separated extendedsecond probe to form a hairpin structure-containing composition; (H)admixing the hairpin structure-containing composition with adepolymerizing amount of an enzyme whose activity is to release one ormore nucleotides from the 3′-terminus of a nucleic acid hybrid to form atreated reaction mixture; (I) maintaining the reaction mixture for atime period sufficient to release 3′-terminal region identifiernucleotides; and (J) analyzing for the presence of released identifiernucleotide to obtain an analytical output, the analytical outputindicating the presence or absence of said predetermined nucleic acidtarget sequence or a specific base within the target sequence.
 143. Themethod according to claim 137 wherein said analytical output is obtainedby luminescence spectroscopy.
 144. The method according to claim 137wherein said analytical output is obtained by fluorescence spectroscopy.145. The method according to claim 139 wherein said released identifiernucleotide includes a fluorescent label.
 146. The method according toclaim 140 wherein said identifier nucleotide is fluorescently labeledafter release from said hybrid.
 147. The method according to claim 137wherein said analytical output is obtained by mass spectrometry. 148.The method according to claim 142 wherein said released identifiernucleotide includes a fluorescent label.
 149. The method according toclaim 143 wherein said identifier nucleotide is fluorescently labeledafter release from said hybrid.
 150. The method according to claim 137wherein said analytical output is obtained by absorbance spectroscopy.151. A process to determine the presence or absence of a predeterminedsingle-stranded nucleic acid target sequence comprising the steps of:(A) providing a depolymerization reaction mixture comprising (i) a pairof first and second complementary nucleic acid probes that form3′-overhangs on both ends of the duplex formed when each of said pair ofcomplementary nucleic acid probes is hybridized with the other, thefirst of said probes being complementary to the nucleic acid targetsequence, (ii) a hybrid between a third probe and the nucleic acidtarget sequence when the nucleic acid target sequence is present in thenucleic acid sample, and (iii) a depolymerizing amount of an enzymewhose activity is to release nucleotides from the 3′-terminus of ahybridized nucleic acid, wherein each of said first and third probes hasan identifier nucleotide in its 3′-terminal region; (B) maintaining thedepolymerization reaction mixture for a time period sufficient to permitthe enzyme to depolymerize the 3′-terminal region of said hybridizedthird probe to release identifier nucleotide and form a first treatedreaction mixture; (C) denaturing the products of the first treatedreaction mixture to form a denatured treated reaction mixture; (D)maintaining the denatured treated reaction mixture for a time periodsufficient to form a second depolymerization reaction mixture thatcomprises (i) a hybrid formed between said first probe and said nucleicacid target sequence when the nucleic acid target sequence is present inthe nucleic acid sample and (ii) a hybrid formed between the3′-terminal-depolymerized third probe and said second nucleic acidprobe, one end of said hybrid having a blunt end or a 5′-overhang aswell as an identifier nucleotide in the 3′-terminal region; (E)depolymerizing hybrids (i) and (ii) of step(D) to release identifiernucleotide from the 3′-terminal regions of said hybrids to form a secondtreated reaction mixture; and (F) analyzing said second treated reactionmixture for the presence of released identifier nucleotide to obtain ananalytical output, the analytical output indicating the presence orabsence of said nucleic acid target sequence.
 152. The process accordingto claim 141 wherein said first and third probes are the same.
 153. Aprocess to determine the presence or absence of a predetermineddouble-stranded nucleic acid target sequence comprising the followingsteps: (A) providing a first reaction mixture comprising (i) first andsecond complementary nucleic acid probes that form 3′-overhangs on bothends of the duplex formed when each of said complementary nucleic acidprobes is hybridized with the other, wherein said each of probes iscomplementary to one or the other strand of the nucleic acid targetsequence and has an identifier nucleotide in its 3′-terminal region,(ii) hybrids between a third and fourth probe and each of the twostrands of the nucleic acid target sequence when the nucleic acid targetsequence is present in the nucleic acid sample, said third and fourthprobes each having an identifier nucleotide in its 3′-terminal regionand (iii) a depolymerizing amount of an enzyme whose activity is torelease nucleotides from the 3′-terminus of a hybridized nucleic acid;(B) maintaining the first reaction mixture for a time period sufficientto permit the enzyme to depolymerize hybridized nucleic acid to releaseidentifier nucleotide from the 3′-terminal region of said hybridizedthird and fourth probes and form a treated first reaction mixture; (C)denaturing the products of the treated first reaction mixture to form adenatured treated reaction mixture; (D) maintaining the denaturedtreated reaction mixture for a time period sufficient to form a secondreaction mixture that comprises a (i) hybrids that lack a 3′-overhangbetween each of the strands of the target nucleic acid and each of thefirst and second probes when the nucleic acid target sequence is presentin the nucleic acid sample, and (ii) hybrids between each of the firstand seconds probes and 3′-terminal region-depolymerized third and fourthprobes, wherein each of said hybrids comprises one end that is blunt orhas a 5′-overhang as well as an identifier nucleotide in the 3′-terminalregion; and (E) depolymerizing the hybrids (i) and (ii) of step (D) torelease identifier nucleotide from the 3′-terminus of said hybridizedprobes to form a second treated reaction mixture; and (F) analyzing saidsecond treated reaction mixture for the presence of released identifiernucleotide to obtain an analytical output, the analytical outputindicating the presence or absence of said nucleic acid target sequence.154. The process according to claim 143 wherein said first and thirdprobes are the same.
 155. The process according to claim 143 whereinsteps A through D are repeated prior to conducting step E.
 156. Anamplification and interrogation process to determine the presence orabsence of a predetermined nucleic acid target sequence comprising thesteps of: (A) providing a ligation reaction solution comprising (i)ligating amount of a ligase, (ii) a nucleic acid sample that may containa predetermined nucleic acid target sequence wherein the nucleic acidtarget sequence has a 3′-portion and a 5′-portion, (iii) an open circleprobe comprising three regions: an open circle probe 3′-terminal region,a linker region, and an open circle probe 5′-terminal region, said opencircle probe further including a detection primer target and anamplification primer target, the amplification primer target beingdownstream of the detection primer target, wherein upon hybridizationbetween the open circle probe and the nucleic acid target sequence, theopen circle probe 3′-terminal region is complementary to a sequence ofthe 3′-portion of said predetermined nucleic acid target sequence, andthe open circle probe 5′-terminal region is complementary to a sequenceof the 5′-portion of said predetermined nucleic acid target sequence,and (iv) optionally further comprising a polymerizing amount of a DNApolymerase and deoxynucleoside triphosphates when the hybridized opencircle probe 3′-terminus is not adjacent and ligatable to the hybridizedopen circle probe 5′-terminus and a gap is present between thosetermini; (B) maintaining the ligation reaction solution for a timeperiod sufficient to permit filling-in of said gap, when present, andligation of the termini of the open circle probe to form a closedcircular probe and a treated ligation reaction solution; (C) admixingsaid closed circular probe with an amplification primer that hybridizeswith said amplification primer target, nucleoside triphosphates, and apolymerizing amount of a DNA polymerase to form a replication reactionmixture; (D) maintaining the replication reaction mixture for a timeperiod sufficient to permit extension of a nucleic acid strand from theamplification primer, wherein the extension product nucleic acid strandcomprises a interrogation target to form a treated replication mixture;(E) admixing an interrogation probe with the treated replicationmixture, wherein the interrogation probe is complementary to saidinterrogation target and comprises an identifier nucleotide in the3′-terminal region; (F) denaturing the treated replication mixture toform a denatured mixture; (G) annealing the denatured mixture to formhybrid between the interrogation probe and the interrogation target whenpresent to form an interrogation solution; (H) admixing a depolymerizingamount of an enzyme whose activity is to release one or more nucleotidesfrom the 3′-terminus of a hybridized nucleic acid probe with theinterrogation solution to form a depolymerization reaction mixture; (I)maintaining the depolymerization reaction mixture for a time periodsufficient to permit the enzyme to depolymerize hybridized nucleic acidand release identifier nucleotide therefrom; and (J) analyzing for thepresence of released identifier nucleotide to obtain an analyticaloutput, the analytical output indicating the presence or absence of saidpredetermined nucleic acid target sequence.
 157. The process accordingto claim 146 wherein the depolymerizing enzyme is thermostable.
 158. Theprocess according to claim 146 wherein free nucleotide triphosphates areseparated from the treated replication mixture prior to step H.
 159. Theprocess according to claim 146 wherein step H occurs before step F. 160.The process according to claim 146 wherein there is a gap presentbetween the termini of the hybridized open circle probe, the portion ofthe predetermined nucleic acid target sequence between the 3′- and5′-termini of the hybridized open circle probe that is opposite said gapcontains three or fewer nucleotides and only nucleoside triphosphatescomplementary to said three or fewer nucleotides are present in saidligation reaction solution.
 161. The process according to claim 146wherein a polymerizing amount of a DNA polymerase and nucleosidetriphosphates are present in said ligation reaction solution.
 162. Theprocess according to claim 146 wherein the open circle probe comprises aplurality of detection primer targets.
 163. The process according toclaim 146 wherein the presence or absence of a plurality ofpredetermined nucleic acid targets is determined using a plurality ofdetection probes comprising different identifier nucleotides.
 164. Theprocess according to claim 153 wherein analysis of the releasedidentifier nucleotides is by mass spectrometry.
 165. An amplificationand interrogation process to determine the presence or absence of apredetermined nucleic acid target sequence having a 3′-portion and a5′-portion comprising the steps of: (A) providing a ligation reactionsolution comprising (i) ligating amount of a ligase, (ii) a nucleic acidsample that may contain a predetermined nucleic acid target sequencewherein the nucleic acid target sequence has a 3′-portion and a5′-portion, (iii) a pair of ligation probes, said ligation probe furtherincluding a detection primer target and an amplification primer target,the amplification primer target being downstream of the detection primertarget, wherein upon hybridization between the open circle probe and thenucleic acid target sequence, the open circle probe 3′-terminal regionis complementary to a sequence of the 3′-portion of said predeterminednucleic acid target sequence, and the open circle probe 5′-terminalregion is complementary to a sequence of the 5′-portion of saidpredetermined nucleic acid target sequence, and (iv) optionally furthercomprising a polymerizing amount of a DNA polymerase and deoxynucleosidetriphosphates when the hybridized open circle probe 3′-terminus is notadjacent and ligatable to the hybridized open circle probe 5′-terminusand a gap is present between those termini; (B) maintaining the ligationreaction solution for a time period sufficient to permit filling-in ofsaid gap, when present, and ligation of the termini of the open circleprobe to form a closed circular probe and a treated ligation reactionsolution; (C) admixing said closed circular probe with an amplificationprimer that hybridizes with said amplification primer target, nucleosidetriphosphates, and a polymerizing amount of a DNA polymerase to form areplication reaction mixture; (D) maintaining the replication reactionmixture for a time period sufficient to permit extension of a nucleicacid strand from the amplification primer, wherein the extension productnucleic acid strand comprises a interrogation target to form a treatedreplication mixture; (E) admixing an interrogation probe with thetreated replication mixture, wherein the interrogation probe iscomplementary to said interrogation target and comprises an identifiernucleotide in the 3′-terminal region; (F) denaturing the treatedreplication mixture to form a denatured mixture; (G) annealing thedenatured mixture to form hybrid between the interrogation probe and theinterrogation target when present to form an interrogation solution; (H)admixing a depolymerizing amount of an enzyme whose activity is torelease one or more nucleotides from the 3′-terminus of a hybridizednucleic acid probe with the interrogation solution to form adepolymerization reaction mixture; (I) maintaining the depolymerizationreaction mixture for a time period sufficient to permit the enzyme todepolymerize hybridized nucleic acid and release identifier nucleotidetherefrom; and (J) analyzing for the presence of released identifiernucleotide to obtain an analytical output, the analytical outputindicating the presence or absence of said predetermined nucleic acidtarget sequence.
 166. The process according to claim 155 wherein thedepolymerizing enzyme is thermostable.
 167. The process according toclaim 155 wherein free nucleotide triphosphates are separated from thetreated replication mixture prior to step H.
 168. The process accordingto claim 155 wherein step H occurs before step F.
 169. The processaccording to claim 155 wherein there is a gap present between thetermini of the hybridized open circle probe, the portion of thepredetermined nucleic acid target sequence between the 3′- and5′-termini of the hybridized open circle probe that is opposite said gapcontains three or fewer nucleotides and only nucleoside triphosphatescomplementary to said three or fewer nucleotides are present in saidligation reaction solution.
 170. The process according to claim 155wherein a polymerizing amount of a DNA polymerase and nucleosidetriphosphates are present in said ligation reaction solution.
 171. Theprocess according to claim 155 wherein the open circle probe comprises aplurality of detection primer targets.
 172. The process according toclaim 155 herein the presence or absence of a plurality of predeterminednucleic acid targets is determined using a plurality of detection probescomprising different identifier nucleotides.
 173. The process accordingto claim 162 wherein analysis of the released identifier nucleotides isby mass spectrometry.
 174. A kit containing a pyrophosphorylation enzymefor use in DNA detection for luminescence spectroscopic analysiscomprising (A) a vessel containing an enzyme capable of catalyzingpyrophosphorolysis; (B) a vessel containing NDPK; and (C) a vesselcontaining ADP.
 175. A kit according to claim 167 further comprising (D)a vessel containing PRPP synthetase.
 176. A kit for determining thepresence or absence of a predetermined nucleic acid target sequence in anucleic acid sample comprising: (A) an enzyme whose activity is torelease one or more nucleotides from the 3′ terminus of a hybridizednucleic acid probe; and (B) at least one nucleic acid probe, saidnucleic acid probe being complementary to nucleic acid target sequence.177. A kit for determining the presence or absence of at least onepredetermined nucleic acid target sequence in a nucleic acid samplecomprising: (A) an enzyme whose activity in the presence ofpyrophosphate is to release identifier nucleotide as a nucleosidetriphosphate from hybridized nucleic acid probe; (B) adenosine 5′diphosphate; (C) pyrophosphate; (D) a nucleoside diphosphate kinase; and(E) at least one nucleic acid probe, said nucleic acid probe beingcomplementary to said predetermined nucleic acid target sequence.
 178. Akit for determining the presence or absence of a predetermined nucleicacid target sequence in a nucleic acid sample comprising: (A) an enzymewhose activity is to release one or more nucleotides from the 3′terminus of a hybridized nucleic acid probe; and (B) instructions foruse.
 179. A composition for use in DNA detection for luminescencespectroscopic analysis comprising an aqueous solution that contains: (A)a purified and isolated enzyme that catalyzes pyrophosphorolysis; (B) apurified and isolated NDPK; and (C) ADP.
 180. The composition accordingaccording to claim 192 further comprising (D) a purified and isolatedPRPP synthetase.
 181. A composition for determining the presence orabsence of a predetermined nucleic acid target sequence in a nucleicacid sample comprising an aqueous solution that contains: (A) a purifiedand isolated enzyme whose activity is to release one or more nucleotidesfrom the 3′ terminus of a hybridized nucleic acid probe; and (B) atleast one nucleic acid probe, said nucleic acid probe beingcomplementary to said predetermined nucleic acid target sequence. 182.The composition according to claim 194 further comprising a purified andisolated nucleoside diphosphate kinase.
 183. The composition accordingto 195, wherein said nucleoside diphosphate kinase is that encoded byPyrococcus furiosis.
 184. A composition of matter for determining thepresence or absence of at least one predetermined nucleic acid targetsequence in a nucleic acid sample comprising an aqueous solution thatcontains: (A) a purified and isolated enzyme whose activity in thepresence of pyrophosphate is to release identifier nucleotide as anucleoside triphosphate from hybridized nucleic acid probe; (B)adenosine 5′ diphosphate; (C) pyrophosphate; (D) a purified and isolatednucleoside diphosphate kinase; and (E) at least one nucleic acid probe,said nucleic acid probe being complementary to said predeterminednucleic acid target sequence.
 185. The composition of matter accordingto claim 197, wherein said purified and isolated enzyme whose activityin the presence of pyrophosphate is to release identifier nucleotides isselected from the group consisting of the Tne triple mutant DNApolymerase, Bst DNA polymerase, Ath DNA polymerase, Tag DNA polymeraseand Tvu DNA polymerase.
 186. The composition of matter according toclaim 197, wherein said purified and isolated nucleoside diphosphatekinase is that encoded by Pyrococcus furiosis.